Characterization of Endogenous Nanoparticles from Roasted Chicken

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Characterization of endogenous nanoparticles from roasted chicken breasts Xunyu Song, Lin Cao, Shuang Cong, Yukun Song, and Mingqian Tan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01988 • Publication Date (Web): 22 Jun 2018 Downloaded from http://pubs.acs.org on June 23, 2018

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Journal of Agricultural and Food Chemistry

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Characterization of endogenous nanoparticles from roasted chicken breasts

2

3

Xunyu Song,1,2 Lin Cao,1,2 Shuang Cong,1,2 Yukun Song,1,2 and Mingqian

4

Tan1,2*

5 6 7

1

School of Food Science and Technology, National Engineering Research Center of

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Seafood, Dalian Polytechnic University, Qinggongyuan1, Ganjingzi District,

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Dalian116034, Liaoning, China

10 11

2

Engineering Research Center of Seafood of Ministry of Education of China,

Dalian116034, Liaoning, China

12 13

*Corresponding author (Tel& Fax: +86-411-86318657, E-mail: [email protected],

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ORCID: 0000000275350035).

15 16 17 18 19

ACS Paragon Plus Environment


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Abstract

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Emergence of endogenous nanoparticles in thermally processed food has aroused

22

much attention due to their unique properties and potential biological impact. The aim

23

of this study was to investigate the presence of fluorescence nanoparticles in roasted

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chicken breasts, elemental composition, physico-chemical properties and their

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molecular interaction with human serum albumin (HSA). Transmission electron

26

microscopy analysis revealed that the foodborne nanoparticles from roasted chicken

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were nearly spherical with an average particle size of 1.7 ± 0.4 nm. The elemental

28

analysis of X-ray photoelectron spectroscopy showed the composition of

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nanoparticles as 47.4% C, 25.8% O and 26.1% N. The fluorescence of HSA was

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quenched by the nanoparticles following a static mode, and the molecular interaction

31

of nanoparticles with HSA was spontaneous (∆G0＜0), where hydrogen bonding and

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van der Waals forces played an important role during HSA-nanoparticles complex

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stabilization through thermodynamic analysis by isothermal titration calorimetry. The

34

principal location of the nanoparticles binding site on HSA was primarily in site I as

35

determined by site-specific marker competition. The conformational of HSA was also

36

changed and ɑ-helical structure decreased in the presence of nanoparticles. Our

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studies revealed that fluorescent nanoparticles were produced after roasting of chicken

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breast at 230 oC for 30 min for the first time. The obtained nanoparticles can interact

39

with HSA in a spontaneous manner, thus providing valuable insight into foodborne

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NPs as well as their effects to human albumin protein.

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Keywords: roasted chicken breasts, fluorescent nanoparticles, human serum albumin,


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thermodynamics, food thermal processing

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1. Introduction

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Chicken meat is one of the most widely accepted foods with high nutritional

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value. As an excellent source of protein, chicken meat has a favourable ratio of

47

unsaturated to saturated fatty acids in its lipids, and provides the necessary vitamins

48

and minerals.1 The roasted chicken breast (RCB) is a popular meat product all over

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the world because of its wonderful flavor and savory taste. The U.S. annual per capita

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consumption of chicken is more than 40 kg.2 In recent years, an increased number of

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studies on RCB has attracted public concern due to the potential health risks

52

associated with high temperature processing.3 These studies focused on the

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unsaturated

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hazardous substance like acrylamide, benzopyrene and heterocyclic amines formed

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during roasting.4-7 These compounds are known to cause several major diseases. From

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a nutritional point of view, the roasting method can cause significant change of

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the safety and nutritional quality of the chicken.4 Actually, Maillard and a series of

58

chemical reactions in the chicken roasting process not only produce the

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above-mentioned harmful substances, but also generate some nanoparticles (NPs)

60

with unknown characteristics and potential health effects.

fatty

acids

content

decreased and formation of a class of

61

Recently, a growing number of studies have described the existence of NPs in the

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daily food containing Maillard reaction products. For example, Sk et al.8 reported the

63

presence of NPs in some high-carbohydrate food in which the preparation processes



64

included heating of the raw material and caramelization reaction. We have reported

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the fluorescent NPs in several commercial beverages,9 which were spherical in shape

66

and well dispersed with a 1-40 nm size and exhibiting an excitation-dependent

67

emission behaviour. In the meantime, we also found a class of carbon nanodots

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collected in roasted fish which were amorphous nanoparticles.10 The above-mentioned

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NPs in food samples are usually very small, highly water soluble and strongly

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fluorescent under light excitation. Therefore, they can pass through the cell membrane

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into the human body and will inevitably contact with the proteins in the blood.

72

However, the thermodynamic properties of molecular interaction between NPs and

73

plasma protein are less studied and our knowledge in this field is still in its infancy.

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Human serum albumin (HSA), the most abundant protein in human blood plasma,

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has complex chemical properties and a series of functional groups that allow

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multifunctional chemical derivatization.11 Thus, HSA has been studied for its

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interaction with nano-sized poly(amidoamine) dendrimers and titanium dioxide as a

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model serum protein.12,13 Xu et al.14 reported the interaction between synthetic carbon

79

nanodots with HSA through hydrophobic and van der Waals forces and their effects

80

on the transportation function. Huang et al.15 studied the in vitro molecular interaction

81

between synthetic nanomaterials and HSA using a spectroscopic method, and found

82

significant binding between them. we recently reported the presence of nanoparticles

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could cause fluorescence quenching of HSA via the electrostatic interaction16. These

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studies demonstrated the possible interactions between many exogenous NPs and

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HSA, causing unpredictable impacts on the function of HSA. These results offered us


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some insight and motivated us to explore the interaction between endogenous NPs

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from food and HSA.

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In this study, the presence of fluorescent NPs in RCB at 230 oC was investigated.

89

The extracted NPs were highly fluorescent, nano-sized, and their physicochemical

90

properties thoroughly characterized. Importantly, we demonstrated the in vitro

91

molecular interaction between the NPs and HSA for the first time using a

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spectroscopic method and thermodynamic analysis technique. In addition, the binding

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site of the NPs from roasted chicken breast and HSA was confirmed by competition

94

experiments to gain a deeper understanding of the interaction, and Fourier transform

95

infrared (FTIR) spectroscopy experiments were used to further prove the changes of

96

the nanoparticles to the HSA structure. The most important observation is that the

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endogenous NPs derived from the roasted chicken breast could bind within site I of

98

HSA and result in a static fluorescence quenching and a conformational change of

99

HSA at the same time. The foodborne NPs from the roasted breasts is likely to attract

100

people’s attention to their influence of human albumin protein.

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2. Materials and Methods

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2.1 Materials

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Chicken breasts was purchased at a local market in Dalian, China. HSA was

104

procured from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

105

Ibuprofen and warfarin were bought from Adamas-beta Co. Ltd. (Shanghai, China).

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Unless otherwise stated, all other chemicals and reagents were of analytical reagent

107

grade and purchased from local suppliers.



108

2.2 Instrumentation

109

Transmission electron microscopy (TEM) analysis was performed using a

110

transmission electron microscope (JEM-2100, JEOL, Tokyo, Japan) at a voltage of

111

200 kV. The molecular weight of the NPs was determined by matrix-assisted laser

112

desorption

113

Autoflex III, Bruker Daltonics, Bremen, Germany), employing 2,5-dihydroxybenzoic

114

acid as the matrix. X-ray diffraction (XRD) data were collected on a diffractometer

115

(XRD-6100, Shimadzu, Kyoto, Japan) with a Cu Kα (λ =1.54060 Å) radiation source

116

from 5 to 60° at 5° min-1 scanning speed. FTIR spectra were obtained by using a FTIR

117

spectrometer (PerkinElmer, Norwalk, CT, U.S.A.). X-ray photoelectron spectroscopy

118

(XPS) spectra of the prepared NPs from RCB were procured using an ESCALAB250

119

XPS system (Thermo VG, Waltham, MA, USA). Absorption spectra were recorded by

120

an ultraviolet-visible (UV-vis) light spectrophotometer (Lambda 35, PerkinElmer,

121

Norwalk, CT, USA), while fluorescence spectra were carried out on a fluorescence

122

spectrometer (F-2700, Hitachi, Tokyo, Japan) at room temperature. Isothermal

123

titration calorimetry (ITC) analyses were obtained by an isothermal titration

124

calorimeter (Affinity ITC SV, TA Instruments, USA). Circular dichroism (CD) spectra

125

were recorded using a JASCO J-1500 CD spectrometer (JASCO, Tokyo, Japan).

126

2.3 Purification of NPs from roasted chicken breast

ionization

time-of-flight

mass

spectrometry (MALDI-TOF-MS,

127

Chicken breasts (190 g) was cut into 1 x 1 x 1 cm pieces after being washed

128

several times with distilled water. Then, they were roasted in an electric heating oven

129

(Rational, SCC-WE-101, Bavaria, Germany) at 230 oC for 30 min. After cooling to


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room temperature, the scorched parts were stripped from the surface of the roasted

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chicken, crushed and extracted with 1200 mL ethanol (100%) at room temperature for

132

36h. After removing the solid precipitate, the ethanol solution was evaporated to

133

remove the solvent. The obtained sticky product was dissolved in 100 mL distilled

134

water and extracted with chloroform (3 × 250 mL). The water phase was subjected to

135

dialysis using a membrane with a molecular weight cutoff of 500 Da against distilled

136

water for 3 days to remove the impurities. The yield of the NPs after lyophilization

137

was approximately 1 g [MALDI-TOF (m/z, [M + H]+): 1033.041 (observed)].

138

2.4 Fluorescence analysis of interaction

139 140 141

To unravel fluorescence quenching mechanism, the fluorescence data were calculated by using the Stern-Volmer equation: F0 = 1 + K q τ 0 [Q ] = 1 + K sv [Q ] F

(1)

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Where F0 and F are the fluorescence intensity values of the protein HSA in the

143

absence and presence of NPs, respectively. KSV is the Stern-Volmer quenching

144

constant, and [Q] is the molar concentration of the quencher NPs. Kq represents the

145

bimolecular quenching rate, and τ0 is the average fluorescence lifetime of the HSA in

146

the absence of quencher, and its value was taken as 10 ns for biomacromolecules.

147 148

For static quenching procedure, the modified Stern-Volmer equation was used for calculating the quenching data.17

149

F0 F 1 1 1 = 0 = + F0 − F ∆F f a K a [Q] f a

150

Where fa represents the fraction of accessible fluorescence, and Ka represents the

151

effective quenching constant for the accessible fluorophore.


(2)


152

3. Results and discussion

153

3.1 Characterization of the NPs extracted from the roasted chicken breast

154

Production of endogenous NPs during food heat processing is a very interesting

155

topic because they may render unknown biological effects after oral absorption. To

156

confirm the presence and formation of NPs after roasting, the extraction products

157

from RCB were dissolved with distilled water, and characterized by TEM using that

158

from raw chicken breast powder (RCBP) as a control. The photographs of raw and

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roasted chicken breast are shown in Figure 1b and their corresponding images of

160

aqueous solution under UV illumination are displayed in Figure 1c. TEM image of the

161

NPs from RCB shows that they were nearly spherical with excellent monodispersity

162

(Figure 1d). The inset represents the high resolution transmission electron microscopy

163

image and no lattice planes were observed, revealing an amorphous structure of NPs

164

as observed previously.18 The size distribution of NPs is relatively narrow falling

165

within 0.6-2.8 nm with an average particle size of 1.7±0.4 nm (Figure 1d and 1e).

166

However, TEM analysis showed that no NPs were formed from the extract of raw

167

chicken breast without roasting (Figure S1). The XRD measurements indicated that

168

there was a broad peak around 2θ =22.6° with a d-spacing of 0.39 nm, which

169

demonstrated that the NPs had an amorphous structure (Figure 1f). This result is

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consistent with that of the high resolution transmission electron microscopy image.

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The mean molecular weight of NPs was 1033 as indicated by the MALDI-TOF-MS

172

result (Figure S2), confirming that the single nanoparticle might have about 74 -CH2-

173

units, or a small number of structures containing carbon, hydrogen, oxygen and


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nitrogen. A reasonable assumption is that the protein and carbohydrates of the chicken

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breasts were decomposed into amorphous carbon-dot-like nanoparticles during the

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roasting process.

177

Surface function compositions of the NPs derived from the roasted chicken

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breasts were further examined by FTIR spectroscopy using RCBP as a control. As

179

depicted in Figure 2a, the NPs and RCBP both show a broad absorption peak in the

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range of 3302-3411 cm-1 ascribed to the O-H stretching vibration of a large number of

181

hydroxyl groups. The large number of hydroxyl groups of NPs may play an important

182

role during the interaction with other proteins. The sharp peaks around 2925-2927

183

cm-1 correspond to the C-H vibrations of methylene. Unlike the RCBP, the NPs show

184

predominance of carboxyl groups and aromatic C=C or CONH2 groups at 1652 cm-1

185

and C-N groups at 1400 cm-1. The peak at 1109 cm-1 indicates the existence of

186

aromatic alkoxy (C-O-C) bonds of the NPs. However, the RCBP shows the

187

characteristic strong absorption peaks at 1538 cm-1, which is assigned to the N-H

188

stretching vibrations.19 The decline of amide functional group strength of NPs might

189

be ascribed to the dissociation of the N-H bond and all of these semaphores reveal

190

that the molecular structures of the NPs mainly contain hydrophilic and polycyclic

191

groups which are connected with the nanostructures.20

192

XPS spectrum in Figure 2b shows two predominant peaks at around 284.8 and

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531.9 eV as well as a weaker peak at 399.6 eV of NPs, which is associated with three

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elements namely C, O and N, respectively. The compositions of the NPs were

195

measured to be 69.87% C, 19.81% O and 9.91% N. Compared with the XPS result of



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RCBP (Figure S3), the NPs from the RCB are probably a kind of N-doped NPs with a

197

higher amount of elemental oxygen after oxidation at high temperature (Table S1).

198

The increase in the content of nitrogen and oxygen of NPs during roasting suggests

199

that the proteins underwent thermal decomposition and air oxidation. The pyrolysis is

200

quite similar to that of carbon dots derived from N-acetylcysteine and diamine via

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hydrothermal treatment.21, 22 The high-resolution XPS spectra further confirmed the

202

presence of different functional groups on the NPs. The C1s spectrum of NPs can be

203

deconvoluted into three peak components with binding energies (Bes) at 284.4, 285.9

204

and 288.1 eV that belong to the C=C, C-O-C/C=N and O-C=C groups, respectively

205

(Figure 2c).23 The N1s core-level spectrum of NPs (Figure 2d) can also be split into

206

three peaks with BEs at 398.9, 399.9 and 400.5, corresponding to the pyridinic

207

nitrogen

208

respectively.24 These data show that the NPs obtained from the RCB are mostly

209

composed of graphitic carbon (sp2) and carbon defects (sp3), and contain an

210

abundance of hydroxy and carbonyl/carboxylate groups at their surfaces.

(C=N-C),

amines

and

amides,

and

H-bonded/protonated

amine,

211

The optical performance of NPs was first analyzed by UV-vis absorption and

212

fluorescence spectroscopy, respectively. Figure 3a shows the typical absorption and

213

fluorescence emission spectra of the NPs in aqueous solution. The UV-vis absorption

214

spectrum exhibits only one weak peak at around 250 nm and extends to 600 nm

215

without noticeable fine structures, demonstrating the presence of aromatic π orbitals.

216

The absorption bands in the region of 250-300 nm are due to the n-π* electronic

217

transitions of the p-π.25 The fluorescence emission spectrum displays a highest value


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of 385 nm under the excitation at 320 nm, and the emission wavelength and intensity

219

show clear excitation wavelength dependency. The bathochromic emission

220

phenomenon is consistent with previous reports on NPs.18 This complex behavior

221

might be associated with various emitting centers and the different surface states

222

present in the suspension and attributed to the different surface states of the NPs.26 In

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short, the decay of fluorescence emission was non-monoexponential for the NPs

224

soliquoid with a lifetime approximately 4.80 ns (Figure 3b). The quantum yield of

225

NPs was 10.8% at the excitation wavelength of 320 nm, by calibrating against quinine

226

sulfate as a reference. The relatively high quantum yield of NPs may originate from

227

the high nitrogen contents that lead to the formation of more trapping excitons under

228

excitation, and it is similar to different NPs found in carbohydrate based food.8, 9

229

It is noteworthy that the fluorescence intensity of NPs could be affected by

230

certain metal ions. As shown in Figure 3c, an equal amount of Ca (II), Mg (I), Mn (I),

231

Co (II), Ni (II) and Fe (II) ions performed a negligible quenching effect. Interestingly,

232

Cu (II) resulted in a weak growth and Fe (III) displayed a significant quenching effect

233

that nearly 90% fluorescence intensity of NPs was declined. The results are attributed

234

to the fact that ions of Cu (II) and Fe (III), etc. are oxidants, and the charges on the NP

235

surface will most likely be captured by these ions. The oxidation-reduction may

236

promote the recombination of non-radiative electrons/holes from the NPs by an

237

electron transfer process.27 It also displayed a pH-dependent fluorescence behavior

238

when the pH ranged from 2 to 11 (Figure 3d). Fluorescence intensity of NPs increases

239

with the increase of pH, and reaches its maximum at pH 5. When the pH is in extreme



240

conditions (at pH 2 or 11), the fluorescence intensity is reduced by about 20 %. Such

241

pH-dependent behaviour is probably due to the dissociation of different functional

242

groups on the surface of the NPs, which may participate in electron-withdrawing and

243

electron-accepting processes.28 Moreover, the influence of NaCl concentration on

244

fluorescence intensity of the NPs was also investigated (Figure S4). When the NaCl

245

concentration increased from 0 to 0.9 mol L-1, the fluorescence intensity of NPs

246

remained unchanged. The bleaching experiment of the NPs in aqueous solution under

247

the UV excitation light showed that the NPs had excellent photostability and no signs

248

of attenuation were displayed for 60 minutes (Figure S5). Thus, these stability

249

properties may require more attention if the NPs can interact with proteins.

250

HSA acts as an important vehicle for transporting hormones, fatty acids, and

251

other compounds and maintains oncotic pressure, among other functions, which

252

possesses three intrinsic fluorophores: tryptophan, tyrosine and phenylalanine

253

residues.29 The variations of fluorescence intensity for HSA could be attributed to the

254

change of amino acid residues. Figure 4a displays the fluorescence spectra of HSA

255

before and after addition of increasing concentrations of NPs derived from RCB. The

256

HSA shows a typical fluorescence emission at 338 nm, originating from Tryptophan.

257

After adding NPs, the fluorescent intensity of HSA decreased significantly,

258

demonstrating that the addition of NPs quenched the intrinsic fluorescence of HSA

259

molecules. An apparent red shift (from 338 to 343 nm) was observed with increasing

260

NPs concentration. This may possibly suggest that the fluorophores of HSA were

261

moved to a more hydrophilic environment after interacting with NPs.30 A linear


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relationship was found with respect to F0/F-1 values and the tested NPs concentration

263

(Figure 4b), in good agreement with the Stern-Volmer equation.

264

Generally speaking, the mechanism of fluorescence quenching can be ascribed to

265

dynamic and static quenching model. Dynamic quenching merely changes the excited

266

states of the fluorophores, while static fluorescence quenching usually results from

267

non-luminous ground-state complexes formed.31 UV-vis absorption spectrum of

268

fluorophore has been considered as one of the reliable tools to illustrate the

269

fluorescence quenching mechanism of HSA and the protein-ligand complex formation

270

in detail.32 Figure 4c shows the UV-vis absorption spectra of HSA, NPs, HSA-NPs

271

and [HSA-NPs]-NPs (the difference of the absorption spectrum between HSA-NPs

272

and NPs). The spectrum of [HSA-NPs]-NPs does not coincide well with the

273

absorption spectrum of HSA at 210-300 nm, indicating that the absorption spectrum

274

of HSA was affected after the addition of NPs. The result revealed a static quenching

275

mechanism of the HSA-NPs system.

276

Time-resolved fluorescence spectrometry is another highly valuable method to

277

straightly illuminate the precise fluorescence quenching mechanism by analyzing the

278

change of the fluorescence lifetime of a protein complex. In time-resolved

279

fluorescence measurements (Figure 4d), the fluorescence decay curves could be best

280

fitted with biexponential equation and the average fluorescence lifetime of HSA alone

281

and HSA in HSA-NPs system were approximately 4.91 and 4.50 ns, respectively.

282

Thus, the observed change is less than 9%, hence the fluorescence lifetime of HSA

283

was considered to be constant. Moreover, the variety of fluorescence lifetime of NPs



284

with the presence of HSA was surveyed to further confirm the fluorescence quenching

285

mechanism. The average fluorescence lifetime of NPs was 4.64 ns in the presence of

286

HSA which was altered only slightly (Figure S6) as compared with that of NPs (4.80

287

ns, Figure 3b). Therefore, we believe that the fluorescence quenching of HSA was

288

induced by the formed complex of HSA-NPs and the liability of static quenching was

289

the major mechanism.

290

3.2 Thermodynamic analysis

291

Over the years, various molecular association mechanisms such as electrostatic,

292

hydrophobic, hydrogen bonding or van der Waals interactions have been proposed for

293

the interaction between protein and NPs.33-36 Specifically, ITC has been extensively

294

used to determine interactions of NPs with HSA and the stoichiometry of proteins

295

bound per nanoparticle derived from the binding curves. Thermodynamic parameters

296

including changes in the enthalpy (∆H), free energy (∆G) and entropy (∆S) can be

297

used to calculate molecular interactions using calorimetric techniques.37, 38 Herein, the

298

sign and dimensions of thermodynamic parameters were employed to provide a

299

comprehensive understanding of the molecular-level interaction mechanism between

300

NPs and HSA. Figure 4e displays the calorimetric profile for the titration of NPs with

301

HSA at ambient temperature. One can see that the correct heat rate of NPs and buffer

302

remained unchanged, while that of the HSA after the addition of NPs increased first

303

then decreased continuously. The heat change per molecule of NPs against the mole

304

ratio (NPs: HSA) at each injection after correcting the heat change for dilution is

305

shown in Figure 4f. The independent site binding model was used to determine the


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thermodynamic parameters, and the fitted parameters were summarized in Table 1.

307

The dissociation constant (Kd) between NPs and HSA was estimated to be 1.29±0.52

308

× 10-4 L mol and the association constant (Ka) was 7.73 × 103 mol L-1 at 298K, which

309

is similar to the previously reported values for different NP-protein complexes.39 This

310

indicates that NPs has binding affinity towards HSA under physiological conditions.

311

The binding stoichiometry (n) per HSA molecule to NP was 1.47±0.11, which is

312

reasonable when considering their sizes. The overall association process between NPs

313

and HSA was spontaneous with a negative standard free energy change (∆G0=-22.20

314

kJ mol-1). Moreover, the process was characterized by a negative standard enthalpy

315

change (∆H0=-3.50±0.33 kJ mol-1) and positive standard entropy change (∆S0=62.71 J

316

mol-1 K-1). Firstly, the process of hydrophobic triggering of the interaction between

317

proteins and ligands is accompanied by a large enthalpy change and a positive entropy

318

change. Therefore, the result cannot be explained by this mechanism. Many cases of

319

relatively larger negative enthalpy change and positive entropy change are usually

320

caused by electrostatic interactions.16 In case of small negative enthalpy change, the

321

binding reaction between HSA and NPs can be ascribed to the interaction of hydrogen

322

bonds or van der Waals. Hence, it is important to carry out further studies at the size

323

and surface charge of NPs and HSA to confirm the major interaction forces between

324

them. The domain I and II of HSA contain most of the negatively charged aspartate

325

and glutamate residues and the isoelectric point of HSA at pH 7.4 is about 4.7.

326

However, the mean zeta potential of NPs of about -4.6 mV is quite small for

327

electrostatic interactions. Moreover, the average particle size of NPs was only 1.7±0.4



328

nm, and the surface of NPs is encapsulated by various functional groups like -CONH2

329

and -OH with high oxygen content which is also beneficial to the hydrogen bond

330

interaction between NPs and HSA.15, 39 This results is different from the previously

331

reported mechanism of interaction between nanoparticles derived from roast duck and

332

proteins,16 and the divergence may be due to the different surface groups of NPs.

333

Taken together, it is suggested that the main mechanism of their association process

334

involves hydrogen bonding and van der Waals force interactions between surface

335

amide and hydroxyl groups (-CONH2 and -OH) of NPs with carboxylate groups

336

(-COO-) of Glu and Asp residues of HSA.

337

3.3 Identification of binding sites of NPs on HSA

338

The binding regions of HSA with other nanostructures are site I (subdomain IIA)

339

and site II (subdomain IIIA), which were reported to be located in the hydrophobic

340

cavities of subdomains. Hence, it is important to further confirm the binding site of

341

HSA with NPs derived from RCB. Warfarin and ibuprofen have usually been regarded

342

as typical site-specific probe for Sudlow’s site I and II of HSA, respectively. Herein,

343

the HSA and warfarin (site I marker) and ibuprofen (site II marker) in equimolar

344

concentration (2 × 10-6 mol L-1) were used for the competitive displacement

345

experiments to identify the NPs binding site on HSA. As shown in Figure 5a, when

346

warfarin was added to HSA, the maximum emission showed a significant red shift,

347

but no change was observed for HSA with the addition of ibuprofen (Figure 5b). To

348

explore the effects of site markers on the binding of HSA-NPs system more directly,

349

the binding constant for the system was analyzed using the modified Stern-Volmer


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equation (Figure 5c). The value of binding constant in the presence of warfarin was

351

obviously variable, whereas a lesser effect of ibuprofen was noted. These results

352

implied that there was a more extensive competition between the NPs and warfarin,

353

and the binding sites for NPs were primarily located in site I (sub-domain IIA) of

354

HSA (Figure 5d).

355

3.4 Conformational change investigation

356

FT-IR spectrometry is a powerful analytical tool to examine the conformational

357

variation in the secondary structure of protein and the interactions with NPs. As

358

shown in Figure 5e, the absorption band of amide I appeared in the region 1600-1650

359

cm-1 (C=O stretch) and amide II band in the region 1500-1550 cm-1 (N-H bending and

360

C-N stretching vibrations), which was compatible with the previous reports.15, 40 The

361

position of the amide I peak shifted slightly from 1646 to 1642 cm-1 and that of the

362

amide II band shifted from 1548 to 1543 cm-1. It is noteworthy that a peak appeared in

363

1581

364

C=O stretching vibration, N-H bending and stretching vibration bands of C-N groups

365

in the polypeptides of HSA were influenced by NPs. Therefore, the secondary

366

structure of the HSA has been induced the rearrangement with the addition of NPs,

367

which is in agreement with the results obtained from ITC experiments.

cm-1 after

the

addition

of

NPs.

The

result

suggested

that

the

368

CD spectroscopy is also regarded as a sensitive and valuable method to study the

369

structure and stability of proteins. It has been reported that the CD spectra of HSA

370

displays two negative absorption bands at 208 and 222 nm, which are contributed by

371

the peptide bond of the α-helix.41 As illustrated in Figure 5f, the relative band



372

intensities of curves 1 to 5 decreased at regular intervals with addition of different

373

concentrations of NPs, indicating a distinct conformational change of the protein with

374

the loss of α-helix content. CD data were computed using Jasco Secondary Structure

375

Estimation (SSE) software and the results are listed in Table 2. The more change of

376

α-helix content is due to the higher concentration of NPs. When the mole ratio of

377

HSA to NPs reaches 1: 100, the content of α-helix decreases from 54.7 to 50.7%. As

378

mentioned above, the binding of NPs may result in the loosening and unfolding of the

379

HSA skeleton and the stronger structural change may affect biological function of

380

protein with the polypeptide chain destabilized.42 These changes clearly demonstrate

381

that NPs were bound with amino acid residues of the main protein chain and wrecked

382

their hydrogen bond network and the suggestion is in good agreement with the results

383

procured by FTIR spectrometry.

384

This work demonstrated the presence of a foodborne NPs specimen generated

385

during the roasting of chicken breast. The strongly fluorescent NPs exhibited an

386

excitation-dependent emission behavior and excellent photostability. Under

387

physiological pH, the intrinsic fluorescence of HSA was quenched by NPs through

388

static mechanism, due to the interaction between HSA and NPs. Thermodynamic

389

parameters revealed that the molecular interaction of NPs with HSA was spontaneous

390

(∆G0＜0), possibly due to the presence of hydrogen bond and van der Waals forces.

391

The binding sites for NPs were basically located in site I of HSA and the secondary

392

structures of HSA changed with the addition of NPs. Thus, this work contributes to

393

drawing attention to the presence of foodborne NPs and their influence on human


Page 18 of 34

Page 19 of 34


394

albumin protein.

395 396

ASSOCIATED CONTENT

397

Supporting Information

398

The Supporting Information is available free of charge on the ACS Publications

399

website at DOI: XXXXXXX.

400

TEM image, XPS spectrum of the extract from raw chicken breast powder without

401

roasting; MALDI-TOF-MS spectral data, photostability, fluorescence decay curve,

402

effect of NaCl solution concentration on the fluorescence intensity of the

403

nanoparticles derived from RCB; composition analysis of the nanoparticles derived

404

from RCB at 230 oC and raw chicken breast powder.

405

406

Acknowledgement

407

This work was supported by the National Key Research and Development

408

Program of China (2017YFD0400103, 2016YFD0400404). We thank Prof. F. Shahidi

409

and Prof. Dayong Zhou for correcting and spelling grammar mistakes.

410

References

411

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412

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fatty acids, vitamin E and selenium on health parameters: a study on male

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applications to bio-imaging. Talanta 2014, 127, 68-74.

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Wang, W.; Liu, W. Nano-carrier for gene delivery and bio-imaging based on

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and Ca2+-dependent potassium channel (BK) gating ring apparatus. J. Biol.

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Chem. 2011, 286, 20701-20709.

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identification of anthocyanin extracted from mulberry fruit and the pigment

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binding properties toward human serum albumin. J. Agric. Food Chem. 2014,

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62, 6813-6819.

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L.; Guo, X. Electrostatic selectivity in protein-nanoparticle interactions.

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Biomacromolecules 2011, 12, 2552-2561.

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poisoning protein function by a hydrophobic nanoparticle. Acs Nano. 2010, 4,

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between bovine serum albumin and amine-functionalized silicon quantum

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dots. Phys. Chem. Chem. Phys. 2014, 16, 8400-8408.


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(36)Sun, W.; Du, Y.; Chen, J.; Kou, J.; Yu, B. Interaction between titanium

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dioxide nanoparticles and human serum albumin revealed by fluorescence

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spectroscopy in the absence of photoactivation. J. Lumin. 2009, 129, 778-783.

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(37)Hartvig, R.A.; Weert, M.V.D.; Østergaard, J.; Jorgensen, L.; Jensen, H.

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Protein adsorption at charged surfaces: the role of electrostatic interactions

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and interfacial charge regulation. Langmuir. 2011, 27, 2634-2643.

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(38)Marangon, M.; Van Sluyter, S.C.; Haynes, P.A.; Waters, E.J. Grape and wine

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proteins: their fractionation by hydrophobic interaction chromatography and

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identification by chromatographic and proteomic analysis. J. Agric. Food

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Chem. 2009, 57, 4415-4425.

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(39)Bhattacharya, A.; Das, S.; Mukherjee, T.K. Insights into the thermodynamics

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of polymer nanodot–human serum albumin association: a spectroscopic and

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calorimetric approach. Langmuir. 2016, 32, 12067-12077.

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(40)Zhang, G.; Wang, L.; Pan, J. Probing the binding of the flavonoid diosmetin

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to human serum albumin by multispectroscopic techniques. J. Agric. Food

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Chem. 2012, 60, 2721-2729.

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(41)Huang, S.; Qiu, H.; Lu, S.; Zhu, F.; Xiao, Q. Study on the molecular

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interaction of graphene quantum dots with human serum albumin: combined

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spectroscopic and electrochemical approaches. J. Hazard. Mater. 2015, 285,

539

18-26.

540 541

(42)Wei, X.L.; Ge, Z.Q. Effect of graphene oxide on conformation and activity of catalase. Carbon 2013, 60, 401-409.



542 543 544

545

Figure captions

546

Figure 1. (a) Schematic illustration of fluorescent NPs derived from roasted chicken

547

breasts. (b) Photograph of the roasted and raw chicken breasts and (c) the NP aqueous

548

solution (left) and water extract (right) of raw chicken breasts under the UV light. (d)

549

TEM image (inset: HR-TEM image, scale bar = 10 nm), (e) corresponding particle

550

size histogram, and (f) XRD pattern of NPs derived from the roasted chicken breasts.

551

Figure 2. (a) FT-IR spectra of raw chicken breast powder (RCBP) and NPs extracted

552

from the roasted chicken breasts, (b) XPS spectrum, and (c) high resolution C1s

553

spectrum of NPs (d) high resolution N1s spectrum of NPs.

554

Figure 3. (a) UV-vis absorption and fluorescence (FL) spectra and (b) fluorescence

555

decay curve of NPs. Effect of metal ions (c) and pH (d) on the fluorescence intensity

556

of the NPs.

557

Figure 4. (a) Fluorescence emission spectra of HSA in the different concentrations of

558

NPs and inserts were the photograph of the HSA and NPs aqueous solution. c (HSA)

559

= 2 × 10 -6 mol L-1, c (NPs), a-h: 0, 0.5, 1, 1.5, 2, 2.5, 3, 4 × 10-4 mol L-1, pH 7.40. (b)

560

Stern-Volmer plots of HSA fluorescence quenched by NPs at 298K. (c) UV-vis

561

absorption spectra of HSA, NPs, HSA-NPs system, and difference of the absorption

562

spectrum between HSA-NPs system and NPs. c (HSA) = 2 × 10-6 mol L-1; c (NPs) = 2


Page 26 of 34

Page 27 of 34


563

× 10-4 mol L-1. (d) Fluorescence decay traces of HSA and HSA-NPs system. τ is

564

fluorescence lifetime of HSA and b is pre-exponential factor, respectively. c (HSA) =

565

2 × 10-6 mol L-1; c (NPs) = 2 × 10-4 mol L-1. (e) Heat flow as a function of time per

566

injection of the NPs in the presence of HSA at 298 K measured by ITC technique. (f)

567

Shows the heat evolved against the molar ratio of (NPs: HSA) at 298 K (black dots).

568

The solid line is the fitted curve.

569

Figure 5. Effect of site marker (a) warfarin and (b) ibuprofen on the fluorescence

570

intensity of the HSA-NPs system (T = 298 K, λex = 280 nm). 1-6: molar ratio of

571

[HAS]/[NPs]=0, 0.5, 1, 1.5, 2 and 2.5, respectively. (c) Modified Stern-Volmer plots

572

for the HSA-NPs system in the absence and presence of warfarin and ibuprofen.

573

c(HSA) = c(warfarin) = c(ibuprofen) = 2 × 10-6 mol L-1; c(NPs)/(104 mol L-1). (d)

574

scheme of identification of binding sites of NPs on HAS. (e) FT-IR spectra of HSA

575

and HSA-NPs system. c (HSA) = 2 × 10-6 mol L-1; c (NPs) = 2 × 10-4 mol L-1. (f) CD

576

spectra of HSA in the presence of NPs at different concentrations of 0, 0.2, 0.4, 1.0,

577

2.0 × 10-4 mol L-1 (curves from 1 to 5); c (HSA) = 2 × 10-6 mol L-1; T= 298 K; pH =

578

7.4.

579 580

Table 1. Thermodynamic parameters for the interaction of CNPs with HSA. Kd (×10-4 L

∆G0 (kJ

∆S0 (J mol-1

mol-1)

mol-1)

K-1)

-3.50 ± 0.33

-22.20

62.71

n

T (K) mol) 298

∆H0 (kJ

1.29 ± 0.52

1.47 ± 0.11



581 582 583 584

Table 2. ɑ-helix and β-sheet contents of HSA with the addition of different

585

concentrations of NPs. Molar ratio [NPs]:[HSA]

ɑ-helix content (%)

0:1

54.7

10:1

54.2

20:1

53.7

50:1

52.8

100:1

50.7

586


Page 28 of 34

Page 29 of 34


a

Roasting Fluorescent NPs

b

c

Raw and roasted chicken breast

d 10 nm

20 nm

f

10 5 0 1.0

587 588

2θ =22.6° ° d=0.39 nm

15

Intensity (a.u.)

Percentage (%)

e

1.5

2.0 Size (nm)

2.5

3.0

10

20

30 40 50 60 2 Theta degree

Figure 1.

589 590 591 592 593 594 595 596 597


70

80


a

b

O1s

O-H 3302

C-H 2927

NPs

C-H 2925

O-H 3411

C-N 1391 C=C N-H 1660 1538

C=C or CONH 1652

C-O-C C-N 1109 1400

3500 3000 2500 2000 1500 1000 500 -1 Wavenumber (cm )

Intensity (a.u.)

C=C 284.4 C-O-C/C=N 285.9 O-C=O 288.1

280

284

288

Intensity (a.u.)

C1s

292

N1s

0

200

d Intensity (a.u.)

Transmittance (a.u.)

RCBP

c

400 600 800 Binding Energy (e.V)

1000

Amines and amides 399.9 Pyridinic N 398.9

396

398

H-bonded or Protonated amine 400.5

400

402

Binding Energy (e.V)

Binding Energy (e.V)

598 599

Page 30 of 34

Figure 2.

600 601 602 603 604 605 606 607 608 609 610 611


404


Absorbance (a.u.) Absorption (a.u.)

Ex

b

300 310 320 330 340 350 360 370 380 390 400 410 420

Intensity (a.u.)

aa

FL Intensity (a.u.)

Page 31 of 34

0

200 250 300 350 400 450 500 550 600

20

Wavelength (nm)

0.8 0.6 0.4 0.2

80

0.8 0.6 0.4 0.2 0.0

--

Bl ac Zn k (I I C ) a( II M ) g( I M I) n( II C ) o( II N ) i(I I Fe ) (I C I) u( I Fe I) (I II )

0.0

612 613

60

d1.0

1.0

FL Intensity (a.u.)

FL Intensity (a.u.)

cc

40 Time (ns)

2

3

4

Ion species

Figure 3.

614 615 616 617 618 619 620 621 622 623 624


5

6

7

pH

8

9

10 11


a

b 0.8

338 nm

298K

(a) FL Intensity (a.u.)

Page 32 of 34

R2=0.998

F0/F-1

0.6

(h) 343 nm

0.4 0.2 4

F0/F-1 =-0.014+1.68×10 [NPs] 0.0

300

350

400

0

450

10

c b

1.0

a

d 1.0

a HSA b NPs c [HSA-NPs] d [HSA-NPs]-[NPs]

Absorbance (a.u.)

Absorbance (a.u.)

1.5

b

c a

d

0.5

d

220

240

260

280

300

Wavelength (nm)

240

t 2 (ns) (b2) (ns)

2.39 (35.37) 6.30 (64.63) 4.91

HSA-NPs 2.17 (42.63) 5.09 (52.99) 4.50

0.6 0.4 0.2

HSA HSA-NPs

270

300

330

360

390

420

0

10

20

30

40

50

60

70

Time (ns)

Wavelength (nm)

e

f

1.6

HSA+NPs Buffer+NPs

1.2 0.8 0.4 0.0 0

800

1600

2400

3200

0.0

Normalized Fit (kJ/mol)

Corrected Heat Rate (µJ/s)

HSA

0.0 210

626

40 -1

Substance t 1 (ns) (b1)

0.8

0.0

625

30

10 [CNPs] (mol L )

Normalized counts

c

20 6

Wavelength (nm)

-0.9 -1.8 -2.7 -3.6 0.0

4000

Time (sec)

0.9 1.8 2.7 Mole Ratio of [NPs]/[HSA]

Figure 4.

627 628 629 630 631 632 633


3.6

Page 33 of 34


a

b

(1)

HSA only (1)

FL intensity (a.u.)

FL intensity (a.u.)

HSA only Warfarin+HSA

(6)

(6)

Warfarin only

300

Ibuprofen only

360

420

300

480

360

F0/(F0-F)

d

Blank Warfarin Ibuprofen

20

420

480

Wavelength (nm)

Wavelength (nm)

c

Ibuprofen+HSA

=Ibuprofen

=Warfarin

15

NPs

10 5

HSA 0.5

1.0

1.5

HSA

2.0

-5

10 [CNPs]-1 (mol L-1)

f-25

1646

-30 HSA

CD (mdeg)

Absorbance (a.u.)

e

1548 1642 C=O

HSA-NPs 1542

-35 -40

1581

-45

1

C-N / N-H

1500

634 635

5

1560

1620

1680

1740

-50 205

210

215

220

Wavelength (nm)

-1 )

Wavenumbers (cm

Figure 5.

636 637 638 639 640 641


225

230


642 643

TOC Roasting

Roasted Chickenq

Chicken

=Warfarin

Purification

NPs water

=Ibuprofen

10 nm

NPs

644

HSA

Fluorescent nanoparticles (NPs)

HSA

20 nm


Page 34 of 34

Characterization of Endogenous Nanoparticles from Roasted Chicken

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