Accepted Manuscript Fluorescence quenching for chloramphenicol detection in milk based on proteinstabilized Au nanoclusters Zhijing Tan, Hua Xu, Gu Li, Xiupei Yang, Martin M.F. Choi PII: DOI: Reference:
S1386-1425(15)00581-8 http://dx.doi.org/10.1016/j.saa.2015.04.109 SAA 13660
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
11 September 2014 26 April 2015 29 April 2015
Please cite this article as: Z. Tan, H. Xu, G. Li, X. Yang, M.M.F. Choi, Fluorescence quenching for chloramphenicol detection in milk based on protein-stabilized Au nanoclusters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.04.109
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1
Fluorescence quenching for chloramphenicol detection in milk based on protein-
2
stabilized Au nanoclusters
3 4 5 6 7 8 9 10 11 12
Zhijing Tana, Hua Xu a, Gu Lia, Xiupei Yanga, *, Martin M.F. Choib,1, ** a
b
College of Chemistry and Chemical Engineering, Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, China West Normal University, Nanchong 637000, P.R. China Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, P.R. China
ABSTRACT
13
In the present study, we report a simple and rapid method for sensitive and selective
14
determination of chloramphenicol (CAP) based on fluorescence of bovine serum albumin-stabilized
15
Au nanoclusters (BSA-AuNCs). The BSA-AuNCs exhibit strong red emission. Upon addition of
16
CAP to BSA-AuNCs, the fluorescence intensity of AuNCs shows a dramatic decrease attributing to
17
the photo-induced electron transfer process from the electrostatically attached CAP to the BSA-
18
AuNCs. The effects of pH, amount of BSA-AuNCs, temperature and reaction time on the detection
19
of chloramphenicol were investigated. Under the optimal conditions, trace amounts of CAP could
20
be detected. The linear working range is 0.10‒70.00 µM with a detection limit 33 nM (S/N = 3). In
21
addition, the proposed method has been successfully applied to the detection of CAP in milk
22
samples and largely improves the application of spectral method for quantitative analysis of CAP.
23 24 25
Keywords: Gold nanoclusters; Chloramphenicol; Bovine serum albumin; Fluorescence
26 27 28 29 30 31 32 33 34
*
Corresponding author at: College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000, PR China. Tel.: +86-817-2568081; fax: +86-817-268067. ** Corresponding author. Fax: +852-34117348. E-mail addresses: [email protected] (X. Yang), [email protected] (M.M.F. Choi). 1 Present address: Acadia Divinity College, Acadia University, 15 University Avenue, Wolfville, Nova Scotia, B4P 2R6, Canada
35
1
36 37
1. Introduction Chloramphenicol
(CAP),
namely
[D(-)-threo-2-dichloro-acetamido-1-p-nitro-phenyl-1,3-
38
propanediol], is a broad spectrum antibiotic that is widely used in animals for the treatment of
39
several infectious diseases because of its effective antibiotic active against gram-positive and gram-
40
negative microorganisms. However, research has shown that it can lead to serious adverse reactions
41
and side effects in humans such as bone marrow suppression, aplastic anemia, cardiovascular
42
collapse [1]. Many countries such as USA, Canada and China ban the use of CAP in the food
43
producing animals. The United States Food and Drug Administration recommended the “minimum
44
required performance limit” (MRPL) of CAP as 0.3 µg·kg-1 for its residues in food products [2].
45
However, CAP is still illegally used in animal farming owing to its accessibility and low cost; thus,
46
its residues have been found in various food samples such as muscle, shrimp, milk and honey [3]. In
47
order to effectively monitor the occurrence of residues of CAP, simple, specific and sensitive
48
analytical methods are required.
49
To date, there are some instrumental analytical methods of CAP in animal food samples, mainly
50
including liquid chromatography (LC) [4,5], liquid chromatography- electrospray ionization tandem
51
mass spectrometry (LC-ESI- MS/MS) [6], gas chromatography-mass spectrometry (GC-MS) [7],
52
capillary zone electrophoresis(CZE) [8], molecular imprinted polymers [9] and electrochemical
53
sensors using variety of unmodified and modified electrodes [10-12]. Among these methods,
54
electrochemical assays are the most widely reported and numerous modified electrodes have been
55
used in CAP detection. But as we all know, their poor repeatability and complex electrode
56
modification process have limited their extensive application in real samples. HPLC and MS are
57
always used together to detect CAP. Although it is selective and sensitive, it always requires
58
expensive equipment, time-consuming extraction and toxic solvents and often involves complex
59
sample pretreatments. CZE also suffer from the defects as HPLC and MS. Therefore, how to
60
develop a simple, costless, fast and reliable assay of CAP has been a challenge for analytical
61
researchers.
2
62
Recently, fluorescence nanoclusters have stimulated extensive interest due to their significant
63
useful optical properties. They have been widely used in biosensors, biomarkers and biomedical
64
imaging [13-20]. Among these fluorescence nanoclusters, gold nanoclusters (AuNCs), an attractive
65
fluorescent probe [21-24], are the most widely used probes in biochemical analysis because of its
66
low toxicity, excellent biocompatibility and stability, good solubility, and excellent luminescence
67
properties [25]. Until now, many kinds of AuNCs with variable fluorescence emissions and
68
quantum yields have been studied. For example, Chen et al. [26] synthesized gold nanoparticles
69
stabilized by papain and applied it in the trace detection of Cu 2+. Su et al. [27] synthesized AuNCs
70
reduced by gallic acid and applied it in the detection of cyromazine. With these fluorescence
71
nanoclusters, various analytical methods have been developed for the detection of biologically
72
environmentally important molecules and ions.
73
In this work, we synthesized water-soluble AuNCs with strong fluorescence, good photostability,
74
and stimuli-responsive properties utilizing bovine serum albumin (BSA) as the protecting ligands.
75
The fluorescence of BSA-AuNCs gradually decreases with the increase in CAP concentration.
76
Herein, we report the development of a fluorescence sensing probe based on BSA-AuNCs for
77
detection of trace amounts of CAP. The analytical feature and the application of the proposed
78
fluorescence quenching method have been fully explored. The major attributes of the proposed
79
method is simple, cost-effective and convenient.
80 81
2. Experimental
82
2.1. Reagents
83
Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O, 99.9%) was purchased from Aldrich
84
(Milwaukee, WI, USA). Bovine serum albumin (BSA, 98.8%) and chloramphenicol (CAP, 99.9%)
85
were obtained from Sigma (St. Louis, MO, USA). Sodium dihydrogen phosphate (NaH2PO4) was
86
obtained from Chongqing Chemical Reagents Co., Ltd. (Chongqing, P.R. China). NaOH was from
87
BeiPei Chemical Reagents Co., Ltd. (Chongqing, P.R. China). NaCl was from Chengdu Kelong
3
88
Chemical Reagents Co., Ltd. (Chengdu, P.R. China). All other reagents of analytical grade were
89
used without further purification.
90 91
2.2. Apparatus
92
Deionized water (18.3 MΩ cm specific resistances) was purified by Human UP 900 Water
93
Purification System (Seoul, Korea). Fourier transform infrared spectra (FTIR) were acquired on a
94
Nicolet 6700 FTIR spectrometer (Thermo Electron Corporation, USA). Absorption spectrum was
95
taken on a Shimadzu UV-2550 UV-vis absorption spectrophotometer (Kyoto, Japan). All
96
fluorescence measurements were made with a Varian Cary Eclipse fluorescence spectrophotometer
97
(Palo Alto, CA, USA) equipped with a 1.0 cm-quartz cell.
98 99
2.3. Synthesis of fluorescent AuNCs
100
BSA-protected gold nanoclusters were prepared using procedures as described previously [28]
101
with some minor modifications. All glassware used in the experiments were washed with freshly
102
prepared aqua regia (HCl:HNO3 3:1 v/v) and rinsed with ultrapure water thoroughly and then oven-
103
dried prior to use. In a typical experiment, into 80 mL deionized water was added 1.6 mL BSA
104
solution (50 mg·mL-1) and 1.6 mL aqueous HAuCl4 solution (10 mM) under vigorous stirring at 37
105
o
C. After 2 min, 1.6 mL NaOH solution (1.0 M) was introduced into the mixture and the reaction
106
was allowed to proceed under vigorous stirring for 12 h. The concentration of BSA-AuNCs formed
107
was 0.20 mM calculated by the number of gold atoms.
108 109
2.4. Fluorescence measurement
110
Into a 5.0 mL calibrated flask was added fixed amounts of BSA-AuNCs, NaH2PO4 buffer
111
solution and CAP. The mixture was incubated 20 min at 25 oC prior to fluorescence measurement.
112
The fluorescence spectra were recorded under excitation at 260 nm and the excitation and emission
113
slits were 10 nm.
4
114 115
2.5. Sample pretreatment
116
Milk samples were bought in a local supermarket and they were pretreated prior to analysis [29].
117
In brief, 1.0 mL milk sample was accurately weighed and then 5.0 mL acetonitrile was added.
118
Afterwards, the mixture vortex for 1 min and pH was adjusted to 2 by addition of few drops of 1 M
119
HCl. The mixture was sonicated in an ultrasonic bath for 20 min and centrifuged at 12,000 rpm for
120
10 min to separate the deposit in the sample matrix. The supernatant was collected and 5.0 mL n-
121
hexane was added. After 10 min shaking, the n-hexane phase was removed and the procedure was
122
repeated for 3 times. Finally, the obtained solution was evaporated to almost dryness at 40 oC. The
123
residue was dissolved in 2. 0 mL deionized water for analysis.
124 125
2.6. Chromatographic analysis
126
The determination of CAP in milk samples was verified by HPLC [30]. The chromatographic
127
analysis was performed on a Waters e2695 Series HPLC system equipped with a quaternary pump,
128
a vacuum degasser, an auto sampler, a thermostated column compartment, a Waters e2996 diode
129
array detector and an Epower data processing system to perform peak purity analyses. Separations
130
were made on a Symmetry® C18 column (150 x 4.6 mm i.d., 5 µm). The mobile phase was a
131
mixture of methanol-water (11:9 v/v) and the flow rate was 0.80 mL min−1. The column
132
temperature was set at 25 oC and the detection wavelength was fixed at 280 nm. 10.0 µL of each
133
sample solution was injected and the data was processed on the Epower Pro data processing system.
134 135
3. Results and discussion
136
3.1. Characterization of the as-prepared BSA-AuNCs
137
The BSA-AuNCs were characterized by UV-visible absorption, fluorescence and infrared
138
spectroscopy. The color of the as-prepared AuNCs solution was light brown under visible light and
139
bright-red under UV light as depicted in the inset of Figure 1A, indicating that highly luminescent
5
140
species were formed. Fig. 1A shows the UV-vis absorption (blue line) and fluorescence emission
141
(red line) spectra of the as-synthesized BSA-AuNCs. The characteristic of the fluorescence spectra
142
of BSA-AuNCs is consistent with those reported in the literature [31]. It can be concluded that the
143
size of BSA-AuNCs is less than 2 nm because the spectrum shows no surface plasmon resonance
144
band. The emission spectrum of AuNCs is in red region around 650 nm upon excitation at 260 nm.
145
Ying et al. [31] proposed that AuNCs were formed in situ by reduction of the entrapped Au ions by
146
the activated BSA molecules, and the BSA stabilized AuNCs have a common magic cluster size of
147
25 Au atoms. Finally, FTIR was used to characterize the attachment of BSA on AuNCs surface as
148
shown in Fig. 1B. The IR spectrum of the BSA-AuNCs is similar to that of BSA, except that the S-
149
H stretching band at 2550 cm-1 of BSA disappears in AuNCs, suggesting the formation of covalent
150
Au-S bond between S-H of BSA and AuNCs. This result indicated that BSA was modified on the
151
surface of AuNCs [32]. Fig. 1
152 153
3.2. The principle of the fluorescence sensor
154
The synthetic strategy for BSA-AuNCs and the principle of CAP sensing are represented in
155
Scheme 1. The as-prepared BSA-AuNCs exhibit strong fluorescence which is considered to arise
156
from intraband transitions of free electrons of the AuNCs [31]. After addition of CAP, the
157
fluorescence intensity of the AuNCs decreases significantly through a photo-induced electron
158
transfer (PET) process. These results suggest that the BSA modified Au nanoclusters could be used
159
as a facile fluorescence quenching sensor for CAP with high sensitivity based on the special
160
interaction between CAP and AuNCs. Scheme 1
161 162
3.3. Optimal conditions for CAP detection
163
In order to investigate the sensitivity, precision and selectivity of the analytical method, the
164
effects of pH, the concentration of BSA-AuNCs, reaction temperature and time of the system were
165
studied.
6
166
3.3.1. Effect of pH
167
The relationship between the fluorescence intensity of BSA-AuNCs and pH in the presence of
168
CAP is shown in Fig. 2(A). In this work, the fluorescence quenching efficiency of CAP on BSA-
169
AuNCs is defined as F0/F where F0 and F are the fluorescence intensities of BSA-AuNCs in the
170
presence and absence of CAP. The fluorescence quenching efficiency (F0/F) of the system increases
171
gradually from pH 4.0 to 5.6 and reaches the maximum at pH 5.6. Further increase in pH results in
172
the decrease in F0/F. The reason may be that chloramphenicol is slightly soluble in water and is
173
stable only in weak acid media. If the pH is too low or too high, the fluorescence quenching
174
efficiency is lower due to the lower solubility of chloramphenicol in water. At pH 5.6, maximum
175
fluorescence quenching efficiency is obtained; thus, 5.6 was chosen as the optimal pH for CAP
176
detection.
177 178
3.3.2. Effect of the amount of BSA-AuNCs
179
Fig. 2(B) displays the effect of the concentration of BSA-AuNCs for detection CAP. When the
180
amount of BSA-AuNCs is too low, the fluorescence quenching efficiency is low because of the
181
limited fluorescent molecules. When the amount of BSA-AuNCs is too high, the fluorescence
182
quenching efficiency decreases because of the self-quenching effect. The fluorescence quenching
183
efficiency is highest at 50 µM BSA-AuNCs and so it was chosen in this work.
184 185
3.3.3. Effect of reaction temperature
186
As reaction temperature is an important factor in determining fluorescence measurement, it was
187
investigated and the results are shown in Fig. 2(C). When the reaction temperature is too high or
188
low, the quenching efficiency is low. The highest quenching efficiency is obtained at 25 oC and so it
189
was selected for this work.
190 191
3.3.4. Effect of reaction time
7
192
Fig. 2(D) displays the effect of reaction time on the fluorescence quenching efficiency of CAP
193
on BSA-AuNPs. The fluorescence quenching efficiency increases with the increase in reaction time
194
(0.0‒20 min). No further increase in fluorescence quenching efficiency after 20 min of reaction time.
195
As such, 20 min was chosen as the optimal reaction time for this work. Fig. 2
196 197
3.4. Selectivity of the proposed method
198
To assess the selectivity of the BSA-AuNCs turn-off fluorescent probe for CAP, the influences
199
of co-existing foreign substances such as L-cysteine, L-serine, phenylalanine, methionine, Na+, Zn2+,
200
Ca2+, K+, Mg2+ were tested and is displayed in Fig. 3. In this study, a potential interferent was
201
initially added at a concentration of 200 times equivalent to CAP. If a significant interference was
202
found, the concentration of this interferent would be gradually reduced until no interference was
203
observed. It was found that most interferents do not produce significant interference on detection of
204
CAP at the concentrations of 100‒200 times of CAP, indicating that the proposed method is highly
205
selective to CAP and it can tolerate high levels of interferents. Fig. 3
206 207
3.5. Fluorescence detection of CAP
208
Fig. 4 shows the change in fluorescence intensity of BSA-AuNCs upon the addition of various
209
concentrations of CAP. The fluorescence intensity decreases with the increase in the concentration
210
of CAP. The inset of Fig. 4 displays the plot of F0/F against the concentration of CAP (CCAP:
211
0.0‒70.0 µM). A good linear relationship was obtained for F0/F and CCAP at 0.10‒70.0 µM: F0/F =
212
1.0719 + 0.0253 CCAP and a correlation coefficient (r) of 0.9996.
213
Fig. 4
214
The limit of detection (LOD) is defined by the equation LOD=3S0/K, where S0 is the standard
215
deviation of blank measurements (n = 11) and K is the slope of calibration graph. Here LOD is 33
216
nM. The repeatability of the proposed method was also evaluated by performing a series of eleven
8
217
repetitive measurements for 10.0 µM CAP and a relative standard deviation (RSD) of 1.52% was
218
obtained. This result suggests that our assay protocol is endowed with good repeatability.
219 220
3.6. Applications
221
To test the feasibility, the proposed method was applied to determine the concentration of CAP
222
in milk samples and the results is depicted in Table 1. CAP was not found in most samples. The
223
recovery tests were done by spiking with CAP at three different concentration levels (0.50, 2.00 and
224
4.00 µM), and then analyzed following the above-described procedures. The concentrations were
225
calculated using the calibration curve. The % recoveries of 98.4‒106 and 97.8‒102% of the intra-
226
day and inter-day analyses were acceptable. The recovery data obtained by this sensor can be used
227
to confirm the accuracy of the method. The precision of the proposed sensor was also evaluated and
228
reported as % RSD of five measurements. The obtained % RSD for the intra-day and inter-day
229
analyses were 1.03‒2.78 and 2.85‒5.38%, respectively. These results confirm that the proposed
230
sensor provided good precision and could potentially be used for the detection of CAP in real
231
samples. Table 1
232 233
In order to compare the analysis of CAP by our proposed method with HPLC, parallel analyses
234
of CAP in non-spiked and spiked milk samples were performed by both methods. Table 2
235
summarizes the results of the determination of CAP in non-spiked and spiked milk samples. The
236
results obtained by our proposed method compare favorably with those obtained by HPLC method,
237
demonstrating that it is very reliable method. Table 2
238 239
3.7. Comparison with other methods
240
A comparison of detection performance between this work and other reported methods in terms
241
of sensitivity and linear range was made and summarizes in Table 3. Our developed assay exhibits a
9
242
LOD and a wider linear range. The RSDs of our proposed method is better than those reported in
243
the literatures. In addition, our method is better than some other methods in terms of the r values.
244
Our LOD is not the lowest but it can be an alternative for determination of CAP in samples as it is
245
simple, convenient and cost-effective. Table 3
246 247
4. Conclusion
248
In this work, the red-emitting BSA-capped AuNCs has been demonstrated to be a selective
249
fluorescence quenching probe for determination of trace amounts of CAP. The present method
250
exhibits a wide detection range, good selectivity, high sensitivity and is immune of coexisting
251
substances interferences. Furthermore, the practical utility of the proposed sensor has been testified
252
for the detection of trace amounts of CAP in milk samples. Our studies have proved that BSA-
253
AuNCs could be a useful luminescence material for practical uses.
254 255
Acknowledgments
256
This work is supported by the National Natural Science Foundation of China (21277109) and the
257
Program for Young Scientific and Technological Innovative Research Team in Sichuan Province
258
(2014TD0020).
259
10
260
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Table 1 Recovery test and precision of the analysis of chloramphenicol in milk samples Sample
Intra-day
Non-spiked Spiked (µΜ)
(µM)
Inter-day
Founda
Recovery
RSD
Founda
Recovery
RSD
(µM)
(%)
(%)
(µM)
(%)
(%)
1
b
ND
0.50
0.49 ± 0.01
98.4
2.78
0.50 ± 0.05
101
3.11
2
NDb
2.00
2.11 ± 0.11
106
1.52
2.04 ± 0.17
102
5.38
3
NDb
4.00
3.99 ± 0.51
99.8
1.03
3.91 ± 0.35
97.8
2.85
314
a
n = 5.
315
b
Not detected.
316
13
317
Table 2 Mean values and standard deviations for the determination of CAP in milk
318
samples by the proposed method and HPLC
Non -spiked Spiked Found (µM)a
Recovery (%)
RSD(%)
(µΜ)
(µM)
This method HPLC
This method HPLC
1
NDb
2.00
2.11 ± 0.11
2.16 ± 0.24
106
108
1.52
2.02
2
NDb
4.00
3.99 ± 0.51
4.02 ± 0.41
99.8
101
1.03
3.71
Sample
319
a
320
b
This method HPLC
n = 5. Not detected.
321
14
322
Table 3 Comparison of the proposed method with other methods for determination of
323
CAP Linear range
Method
(µΜ)
R
LOD
RSD
(µΜ)
(%)
Ref.
Electrochemical detection
2.00‒80.0
0.9985
0.59
2.84
[11]
Gold nanocatalyst-based immunosensing
0.0003‒0.3
0.9972
0.00009
‒
[33]
Square wave voltammetry
0.10‒10.0
‒
0.047
‒
[29]
Luminol functionalized silver nanoprobe
0.03‒3.0
0.9970
0.024
‒
[34]
Chemiluminescent immunosensor
0.01‒100.0
‒
0.01
‒
[35]
High-performance liquid chromatography
0.002‒1.5
0.9995
0.0003
4.50
[5]
FIA with amperometric detection
50.0‒1000
‒
44.0
‒
[36]
0.01‒6.0
0.9990
0.005
‒
[12]
‒
‒
0.0001
‒
[37]
with
5.0‒1000
0.9996
0.91
‒
[9]
Molecularly imprinted membrane- based
0.01‒12.0
0.9870
0.002
2.50
[10]
mass
0.03‒3.0
0.9998
0.022
‒
[38]
microextraction-liquid
0.10‒3.0
0.9997
0.1
‒
[39]
0.10‒70.00
0.9998
0.033
1.52
This work
Voltammetry Surface plasmon resonance assay Capillary
zone
electrophoresis
amperometric detection
sensor Liquid
chromatography-tandem
spectrometry Solid
phase
chromatography Fluorescence quenching based on BSAAuNCs 324 325
15
326
Scheme 1 Scheme of the synthetic strategy for BSA-AuNCs and the principle of CA
327
sensing.
328 329
330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355
Figure Captions Fig. 1. (A) Absorbance and fluorescence emission spectra of the BSA-AuNCs. Inset: photographic images under visible light (left) and ultraviolet light (right) of the BSAAuNCs. (B) Infrared spectra of (a) BSA and (b) BSA-AuNCs.
Fig. 2. Effect of (A) pH, (B) the amount of BSA-AuNCs, (C) reaction temperature and (D) reaction time on the fluorescence intensity of BSA-AuNCs-CAP system. F0 and F are the fluorescence intensity of BSA-AuNCs in the absence and presence of 2.50 µM CAP in phosphate buffer solution (30 mM, pH = 5.6).
Fig. 3. Effect of potentially interfering substances. CAP: 30.00 µΜ; glycine, histidine, L-cysteine, lysine, phenylalanine, aspartic acid, Na+, K+, Ca2+, and Mg2+: 6.00 mM and others: 3.00 mM. The inset displays the fluorescence spectra for potentially interfering substances. 50 µM AuNCs in phosphate buffer solution (30 mM, pH = 5.6) is used
Fig. 4. The change in fluorescence intensity of BSA-AuNCs upon the addition of various concentrations of CAP. The concentrations of CAP are: (a) 0.00, (b) 0.10, (c) 1.25, (d) 2.50, (e) 5.00, (f) 10.00, (g) 20.00, (h) 30.00, (i) 40.00, (j) 50.00, (k) 60.00, and (l) 70.00 µM. The inset displays the linear plot of F0/F against CCAP concentration of CAP. 50 µM AuNCs in phosphate buffer solution (30 mM, pH = 5.6) is used.
17
Fig. 1.
356 357 358 0.8 A
0.4 400
0.2 200
0.0 0
400
500
600
Wavelength (nm)
700
800
(b) BSA-AuNCs Transmittance (%)
600
Fluorescence Intensity (a.u.)
Absorbance (a.u.)
0.6
300
B
800
4000
(a) BSA
3500
3000
2500
2000
1500 -1 Wavenumber (cm )
1000
500
18
Fig. 2.
359
1.16
1.15
A
1.14
1.14
1.12
1.13
1.10
F0/F
F0/F
B
1.08
1.12 1.11
1.06 1.10
1.04
4
5
6
7
0.00
8
0.02
0.06
0.08
0.10
CBSA-AuNPs (mM)
pH
1.16
0.04
1.16
C
D
1.14
1.14
1.12 1.10 F0/F
F0/F
1.12 1.10
1.08 1.06
1.08
1.04
1.06
1.02
0
10
20
30
40
Temperature ( C) O
50
0
5
10
15
20
25
Time (min)
360 361
19
Fig. 3.
362
1.8 1.7 1.6
F0/F
1.5 1.4 1.3 1.2 1.1
Fluorescence intensity (a.u.)
500
Gly, His, Lys, Ser, Met, Val, Leu, Hcy, Cys, Phe, Asp, Na+, Zn2+, K+,
Blank
400
Ca2+, Mg2+ CAP
300 200 100 550
600
650
700
750
800
Wavelength (nm)
1.0
Bl
an k G ly H is Ly s Ph Ae sp Se Mr et V al Le u H cy C ys N + a K+ C 2 a + Zn 2 + M g 2+ C A P
BlankGly His Lys PheAsp Ser Met Val Leu Hcy CysNa+ K+Ca2+ Zn2+ Mg2+CAP
363
20
Fig. 4.
364
F0/F
Fluorescence intensity (a.u.)
2.8 F0/F=0.0253C+1.0719 R2=0.9996 2.4
a
600
500
2.0 1.6 1.2
400
0
10 20 30 40 50 60 70 80 CCAP (µM)
µM
l
300
200
100
0 550
600
650
700
Wavelength (nm) 365 366
750
800
367
Highlights
368
Bovine serum albumin stabilized Au nanoclusters were synthesized in aqueous solution.
369
The protein-stabilized water-soluble Au nanoclusters showed well fluorescence properties.
370
A novel fluorescent sensor for chloramphenicol was proposed.
371 372
22
373
Graphical abstract
374
23
S1386-1425(15)00581-8 http://dx.doi.org/10.1016/j.saa.2015.04.109 SAA 13660
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
11 September 2014 26 April 2015 29 April 2015
Please cite this article as: Z. Tan, H. Xu, G. Li, X. Yang, M.M.F. Choi, Fluorescence quenching for chloramphenicol detection in milk based on protein-stabilized Au nanoclusters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.04.109
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Fluorescence quenching for chloramphenicol detection in milk based on protein-
2
stabilized Au nanoclusters
3 4 5 6 7 8 9 10 11 12
Zhijing Tana, Hua Xu a, Gu Lia, Xiupei Yanga, *, Martin M.F. Choib,1, ** a
b
College of Chemistry and Chemical Engineering, Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province, China West Normal University, Nanchong 637000, P.R. China Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR, P.R. China
ABSTRACT
13
In the present study, we report a simple and rapid method for sensitive and selective
14
determination of chloramphenicol (CAP) based on fluorescence of bovine serum albumin-stabilized
15
Au nanoclusters (BSA-AuNCs). The BSA-AuNCs exhibit strong red emission. Upon addition of
16
CAP to BSA-AuNCs, the fluorescence intensity of AuNCs shows a dramatic decrease attributing to
17
the photo-induced electron transfer process from the electrostatically attached CAP to the BSA-
18
AuNCs. The effects of pH, amount of BSA-AuNCs, temperature and reaction time on the detection
19
of chloramphenicol were investigated. Under the optimal conditions, trace amounts of CAP could
20
be detected. The linear working range is 0.10‒70.00 µM with a detection limit 33 nM (S/N = 3). In
21
addition, the proposed method has been successfully applied to the detection of CAP in milk
22
samples and largely improves the application of spectral method for quantitative analysis of CAP.
23 24 25
Keywords: Gold nanoclusters; Chloramphenicol; Bovine serum albumin; Fluorescence
26 27 28 29 30 31 32 33 34
*
Corresponding author at: College of Chemistry and Chemical Engineering, China West Normal University, Nanchong 637000, PR China. Tel.: +86-817-2568081; fax: +86-817-268067. ** Corresponding author. Fax: +852-34117348. E-mail addresses: [email protected] (X. Yang), [email protected] (M.M.F. Choi). 1 Present address: Acadia Divinity College, Acadia University, 15 University Avenue, Wolfville, Nova Scotia, B4P 2R6, Canada
35
1
36 37
1. Introduction Chloramphenicol
(CAP),
namely
[D(-)-threo-2-dichloro-acetamido-1-p-nitro-phenyl-1,3-
38
propanediol], is a broad spectrum antibiotic that is widely used in animals for the treatment of
39
several infectious diseases because of its effective antibiotic active against gram-positive and gram-
40
negative microorganisms. However, research has shown that it can lead to serious adverse reactions
41
and side effects in humans such as bone marrow suppression, aplastic anemia, cardiovascular
42
collapse [1]. Many countries such as USA, Canada and China ban the use of CAP in the food
43
producing animals. The United States Food and Drug Administration recommended the “minimum
44
required performance limit” (MRPL) of CAP as 0.3 µg·kg-1 for its residues in food products [2].
45
However, CAP is still illegally used in animal farming owing to its accessibility and low cost; thus,
46
its residues have been found in various food samples such as muscle, shrimp, milk and honey [3]. In
47
order to effectively monitor the occurrence of residues of CAP, simple, specific and sensitive
48
analytical methods are required.
49
To date, there are some instrumental analytical methods of CAP in animal food samples, mainly
50
including liquid chromatography (LC) [4,5], liquid chromatography- electrospray ionization tandem
51
mass spectrometry (LC-ESI- MS/MS) [6], gas chromatography-mass spectrometry (GC-MS) [7],
52
capillary zone electrophoresis(CZE) [8], molecular imprinted polymers [9] and electrochemical
53
sensors using variety of unmodified and modified electrodes [10-12]. Among these methods,
54
electrochemical assays are the most widely reported and numerous modified electrodes have been
55
used in CAP detection. But as we all know, their poor repeatability and complex electrode
56
modification process have limited their extensive application in real samples. HPLC and MS are
57
always used together to detect CAP. Although it is selective and sensitive, it always requires
58
expensive equipment, time-consuming extraction and toxic solvents and often involves complex
59
sample pretreatments. CZE also suffer from the defects as HPLC and MS. Therefore, how to
60
develop a simple, costless, fast and reliable assay of CAP has been a challenge for analytical
61
researchers.
2
62
Recently, fluorescence nanoclusters have stimulated extensive interest due to their significant
63
useful optical properties. They have been widely used in biosensors, biomarkers and biomedical
64
imaging [13-20]. Among these fluorescence nanoclusters, gold nanoclusters (AuNCs), an attractive
65
fluorescent probe [21-24], are the most widely used probes in biochemical analysis because of its
66
low toxicity, excellent biocompatibility and stability, good solubility, and excellent luminescence
67
properties [25]. Until now, many kinds of AuNCs with variable fluorescence emissions and
68
quantum yields have been studied. For example, Chen et al. [26] synthesized gold nanoparticles
69
stabilized by papain and applied it in the trace detection of Cu 2+. Su et al. [27] synthesized AuNCs
70
reduced by gallic acid and applied it in the detection of cyromazine. With these fluorescence
71
nanoclusters, various analytical methods have been developed for the detection of biologically
72
environmentally important molecules and ions.
73
In this work, we synthesized water-soluble AuNCs with strong fluorescence, good photostability,
74
and stimuli-responsive properties utilizing bovine serum albumin (BSA) as the protecting ligands.
75
The fluorescence of BSA-AuNCs gradually decreases with the increase in CAP concentration.
76
Herein, we report the development of a fluorescence sensing probe based on BSA-AuNCs for
77
detection of trace amounts of CAP. The analytical feature and the application of the proposed
78
fluorescence quenching method have been fully explored. The major attributes of the proposed
79
method is simple, cost-effective and convenient.
80 81
2. Experimental
82
2.1. Reagents
83
Hydrogen tetrachloroaurate(III) trihydrate (HAuCl4·3H2O, 99.9%) was purchased from Aldrich
84
(Milwaukee, WI, USA). Bovine serum albumin (BSA, 98.8%) and chloramphenicol (CAP, 99.9%)
85
were obtained from Sigma (St. Louis, MO, USA). Sodium dihydrogen phosphate (NaH2PO4) was
86
obtained from Chongqing Chemical Reagents Co., Ltd. (Chongqing, P.R. China). NaOH was from
87
BeiPei Chemical Reagents Co., Ltd. (Chongqing, P.R. China). NaCl was from Chengdu Kelong
3
88
Chemical Reagents Co., Ltd. (Chengdu, P.R. China). All other reagents of analytical grade were
89
used without further purification.
90 91
2.2. Apparatus
92
Deionized water (18.3 MΩ cm specific resistances) was purified by Human UP 900 Water
93
Purification System (Seoul, Korea). Fourier transform infrared spectra (FTIR) were acquired on a
94
Nicolet 6700 FTIR spectrometer (Thermo Electron Corporation, USA). Absorption spectrum was
95
taken on a Shimadzu UV-2550 UV-vis absorption spectrophotometer (Kyoto, Japan). All
96
fluorescence measurements were made with a Varian Cary Eclipse fluorescence spectrophotometer
97
(Palo Alto, CA, USA) equipped with a 1.0 cm-quartz cell.
98 99
2.3. Synthesis of fluorescent AuNCs
100
BSA-protected gold nanoclusters were prepared using procedures as described previously [28]
101
with some minor modifications. All glassware used in the experiments were washed with freshly
102
prepared aqua regia (HCl:HNO3 3:1 v/v) and rinsed with ultrapure water thoroughly and then oven-
103
dried prior to use. In a typical experiment, into 80 mL deionized water was added 1.6 mL BSA
104
solution (50 mg·mL-1) and 1.6 mL aqueous HAuCl4 solution (10 mM) under vigorous stirring at 37
105
o
C. After 2 min, 1.6 mL NaOH solution (1.0 M) was introduced into the mixture and the reaction
106
was allowed to proceed under vigorous stirring for 12 h. The concentration of BSA-AuNCs formed
107
was 0.20 mM calculated by the number of gold atoms.
108 109
2.4. Fluorescence measurement
110
Into a 5.0 mL calibrated flask was added fixed amounts of BSA-AuNCs, NaH2PO4 buffer
111
solution and CAP. The mixture was incubated 20 min at 25 oC prior to fluorescence measurement.
112
The fluorescence spectra were recorded under excitation at 260 nm and the excitation and emission
113
slits were 10 nm.
4
114 115
2.5. Sample pretreatment
116
Milk samples were bought in a local supermarket and they were pretreated prior to analysis [29].
117
In brief, 1.0 mL milk sample was accurately weighed and then 5.0 mL acetonitrile was added.
118
Afterwards, the mixture vortex for 1 min and pH was adjusted to 2 by addition of few drops of 1 M
119
HCl. The mixture was sonicated in an ultrasonic bath for 20 min and centrifuged at 12,000 rpm for
120
10 min to separate the deposit in the sample matrix. The supernatant was collected and 5.0 mL n-
121
hexane was added. After 10 min shaking, the n-hexane phase was removed and the procedure was
122
repeated for 3 times. Finally, the obtained solution was evaporated to almost dryness at 40 oC. The
123
residue was dissolved in 2. 0 mL deionized water for analysis.
124 125
2.6. Chromatographic analysis
126
The determination of CAP in milk samples was verified by HPLC [30]. The chromatographic
127
analysis was performed on a Waters e2695 Series HPLC system equipped with a quaternary pump,
128
a vacuum degasser, an auto sampler, a thermostated column compartment, a Waters e2996 diode
129
array detector and an Epower data processing system to perform peak purity analyses. Separations
130
were made on a Symmetry® C18 column (150 x 4.6 mm i.d., 5 µm). The mobile phase was a
131
mixture of methanol-water (11:9 v/v) and the flow rate was 0.80 mL min−1. The column
132
temperature was set at 25 oC and the detection wavelength was fixed at 280 nm. 10.0 µL of each
133
sample solution was injected and the data was processed on the Epower Pro data processing system.
134 135
3. Results and discussion
136
3.1. Characterization of the as-prepared BSA-AuNCs
137
The BSA-AuNCs were characterized by UV-visible absorption, fluorescence and infrared
138
spectroscopy. The color of the as-prepared AuNCs solution was light brown under visible light and
139
bright-red under UV light as depicted in the inset of Figure 1A, indicating that highly luminescent
5
140
species were formed. Fig. 1A shows the UV-vis absorption (blue line) and fluorescence emission
141
(red line) spectra of the as-synthesized BSA-AuNCs. The characteristic of the fluorescence spectra
142
of BSA-AuNCs is consistent with those reported in the literature [31]. It can be concluded that the
143
size of BSA-AuNCs is less than 2 nm because the spectrum shows no surface plasmon resonance
144
band. The emission spectrum of AuNCs is in red region around 650 nm upon excitation at 260 nm.
145
Ying et al. [31] proposed that AuNCs were formed in situ by reduction of the entrapped Au ions by
146
the activated BSA molecules, and the BSA stabilized AuNCs have a common magic cluster size of
147
25 Au atoms. Finally, FTIR was used to characterize the attachment of BSA on AuNCs surface as
148
shown in Fig. 1B. The IR spectrum of the BSA-AuNCs is similar to that of BSA, except that the S-
149
H stretching band at 2550 cm-1 of BSA disappears in AuNCs, suggesting the formation of covalent
150
Au-S bond between S-H of BSA and AuNCs. This result indicated that BSA was modified on the
151
surface of AuNCs [32]. Fig. 1
152 153
3.2. The principle of the fluorescence sensor
154
The synthetic strategy for BSA-AuNCs and the principle of CAP sensing are represented in
155
Scheme 1. The as-prepared BSA-AuNCs exhibit strong fluorescence which is considered to arise
156
from intraband transitions of free electrons of the AuNCs [31]. After addition of CAP, the
157
fluorescence intensity of the AuNCs decreases significantly through a photo-induced electron
158
transfer (PET) process. These results suggest that the BSA modified Au nanoclusters could be used
159
as a facile fluorescence quenching sensor for CAP with high sensitivity based on the special
160
interaction between CAP and AuNCs. Scheme 1
161 162
3.3. Optimal conditions for CAP detection
163
In order to investigate the sensitivity, precision and selectivity of the analytical method, the
164
effects of pH, the concentration of BSA-AuNCs, reaction temperature and time of the system were
165
studied.
6
166
3.3.1. Effect of pH
167
The relationship between the fluorescence intensity of BSA-AuNCs and pH in the presence of
168
CAP is shown in Fig. 2(A). In this work, the fluorescence quenching efficiency of CAP on BSA-
169
AuNCs is defined as F0/F where F0 and F are the fluorescence intensities of BSA-AuNCs in the
170
presence and absence of CAP. The fluorescence quenching efficiency (F0/F) of the system increases
171
gradually from pH 4.0 to 5.6 and reaches the maximum at pH 5.6. Further increase in pH results in
172
the decrease in F0/F. The reason may be that chloramphenicol is slightly soluble in water and is
173
stable only in weak acid media. If the pH is too low or too high, the fluorescence quenching
174
efficiency is lower due to the lower solubility of chloramphenicol in water. At pH 5.6, maximum
175
fluorescence quenching efficiency is obtained; thus, 5.6 was chosen as the optimal pH for CAP
176
detection.
177 178
3.3.2. Effect of the amount of BSA-AuNCs
179
Fig. 2(B) displays the effect of the concentration of BSA-AuNCs for detection CAP. When the
180
amount of BSA-AuNCs is too low, the fluorescence quenching efficiency is low because of the
181
limited fluorescent molecules. When the amount of BSA-AuNCs is too high, the fluorescence
182
quenching efficiency decreases because of the self-quenching effect. The fluorescence quenching
183
efficiency is highest at 50 µM BSA-AuNCs and so it was chosen in this work.
184 185
3.3.3. Effect of reaction temperature
186
As reaction temperature is an important factor in determining fluorescence measurement, it was
187
investigated and the results are shown in Fig. 2(C). When the reaction temperature is too high or
188
low, the quenching efficiency is low. The highest quenching efficiency is obtained at 25 oC and so it
189
was selected for this work.
190 191
3.3.4. Effect of reaction time
7
192
Fig. 2(D) displays the effect of reaction time on the fluorescence quenching efficiency of CAP
193
on BSA-AuNPs. The fluorescence quenching efficiency increases with the increase in reaction time
194
(0.0‒20 min). No further increase in fluorescence quenching efficiency after 20 min of reaction time.
195
As such, 20 min was chosen as the optimal reaction time for this work. Fig. 2
196 197
3.4. Selectivity of the proposed method
198
To assess the selectivity of the BSA-AuNCs turn-off fluorescent probe for CAP, the influences
199
of co-existing foreign substances such as L-cysteine, L-serine, phenylalanine, methionine, Na+, Zn2+,
200
Ca2+, K+, Mg2+ were tested and is displayed in Fig. 3. In this study, a potential interferent was
201
initially added at a concentration of 200 times equivalent to CAP. If a significant interference was
202
found, the concentration of this interferent would be gradually reduced until no interference was
203
observed. It was found that most interferents do not produce significant interference on detection of
204
CAP at the concentrations of 100‒200 times of CAP, indicating that the proposed method is highly
205
selective to CAP and it can tolerate high levels of interferents. Fig. 3
206 207
3.5. Fluorescence detection of CAP
208
Fig. 4 shows the change in fluorescence intensity of BSA-AuNCs upon the addition of various
209
concentrations of CAP. The fluorescence intensity decreases with the increase in the concentration
210
of CAP. The inset of Fig. 4 displays the plot of F0/F against the concentration of CAP (CCAP:
211
0.0‒70.0 µM). A good linear relationship was obtained for F0/F and CCAP at 0.10‒70.0 µM: F0/F =
212
1.0719 + 0.0253 CCAP and a correlation coefficient (r) of 0.9996.
213
Fig. 4
214
The limit of detection (LOD) is defined by the equation LOD=3S0/K, where S0 is the standard
215
deviation of blank measurements (n = 11) and K is the slope of calibration graph. Here LOD is 33
216
nM. The repeatability of the proposed method was also evaluated by performing a series of eleven
8
217
repetitive measurements for 10.0 µM CAP and a relative standard deviation (RSD) of 1.52% was
218
obtained. This result suggests that our assay protocol is endowed with good repeatability.
219 220
3.6. Applications
221
To test the feasibility, the proposed method was applied to determine the concentration of CAP
222
in milk samples and the results is depicted in Table 1. CAP was not found in most samples. The
223
recovery tests were done by spiking with CAP at three different concentration levels (0.50, 2.00 and
224
4.00 µM), and then analyzed following the above-described procedures. The concentrations were
225
calculated using the calibration curve. The % recoveries of 98.4‒106 and 97.8‒102% of the intra-
226
day and inter-day analyses were acceptable. The recovery data obtained by this sensor can be used
227
to confirm the accuracy of the method. The precision of the proposed sensor was also evaluated and
228
reported as % RSD of five measurements. The obtained % RSD for the intra-day and inter-day
229
analyses were 1.03‒2.78 and 2.85‒5.38%, respectively. These results confirm that the proposed
230
sensor provided good precision and could potentially be used for the detection of CAP in real
231
samples. Table 1
232 233
In order to compare the analysis of CAP by our proposed method with HPLC, parallel analyses
234
of CAP in non-spiked and spiked milk samples were performed by both methods. Table 2
235
summarizes the results of the determination of CAP in non-spiked and spiked milk samples. The
236
results obtained by our proposed method compare favorably with those obtained by HPLC method,
237
demonstrating that it is very reliable method. Table 2
238 239
3.7. Comparison with other methods
240
A comparison of detection performance between this work and other reported methods in terms
241
of sensitivity and linear range was made and summarizes in Table 3. Our developed assay exhibits a
9
242
LOD and a wider linear range. The RSDs of our proposed method is better than those reported in
243
the literatures. In addition, our method is better than some other methods in terms of the r values.
244
Our LOD is not the lowest but it can be an alternative for determination of CAP in samples as it is
245
simple, convenient and cost-effective. Table 3
246 247
4. Conclusion
248
In this work, the red-emitting BSA-capped AuNCs has been demonstrated to be a selective
249
fluorescence quenching probe for determination of trace amounts of CAP. The present method
250
exhibits a wide detection range, good selectivity, high sensitivity and is immune of coexisting
251
substances interferences. Furthermore, the practical utility of the proposed sensor has been testified
252
for the detection of trace amounts of CAP in milk samples. Our studies have proved that BSA-
253
AuNCs could be a useful luminescence material for practical uses.
254 255
Acknowledgments
256
This work is supported by the National Natural Science Foundation of China (21277109) and the
257
Program for Young Scientific and Technological Innovative Research Team in Sichuan Province
258
(2014TD0020).
259
10
260
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312 313
Table 1 Recovery test and precision of the analysis of chloramphenicol in milk samples Sample
Intra-day
Non-spiked Spiked (µΜ)
(µM)
Inter-day
Founda
Recovery
RSD
Founda
Recovery
RSD
(µM)
(%)
(%)
(µM)
(%)
(%)
1
b
ND
0.50
0.49 ± 0.01
98.4
2.78
0.50 ± 0.05
101
3.11
2
NDb
2.00
2.11 ± 0.11
106
1.52
2.04 ± 0.17
102
5.38
3
NDb
4.00
3.99 ± 0.51
99.8
1.03
3.91 ± 0.35
97.8
2.85
314
a
n = 5.
315
b
Not detected.
316
13
317
Table 2 Mean values and standard deviations for the determination of CAP in milk
318
samples by the proposed method and HPLC
Non -spiked Spiked Found (µM)a
Recovery (%)
RSD(%)
(µΜ)
(µM)
This method HPLC
This method HPLC
1
NDb
2.00
2.11 ± 0.11
2.16 ± 0.24
106
108
1.52
2.02
2
NDb
4.00
3.99 ± 0.51
4.02 ± 0.41
99.8
101
1.03
3.71
Sample
319
a
320
b
This method HPLC
n = 5. Not detected.
321
14
322
Table 3 Comparison of the proposed method with other methods for determination of
323
CAP Linear range
Method
(µΜ)
R
LOD
RSD
(µΜ)
(%)
Ref.
Electrochemical detection
2.00‒80.0
0.9985
0.59
2.84
[11]
Gold nanocatalyst-based immunosensing
0.0003‒0.3
0.9972
0.00009
‒
[33]
Square wave voltammetry
0.10‒10.0
‒
0.047
‒
[29]
Luminol functionalized silver nanoprobe
0.03‒3.0
0.9970
0.024
‒
[34]
Chemiluminescent immunosensor
0.01‒100.0
‒
0.01
‒
[35]
High-performance liquid chromatography
0.002‒1.5
0.9995
0.0003
4.50
[5]
FIA with amperometric detection
50.0‒1000
‒
44.0
‒
[36]
0.01‒6.0
0.9990
0.005
‒
[12]
‒
‒
0.0001
‒
[37]
with
5.0‒1000
0.9996
0.91
‒
[9]
Molecularly imprinted membrane- based
0.01‒12.0
0.9870
0.002
2.50
[10]
mass
0.03‒3.0
0.9998
0.022
‒
[38]
microextraction-liquid
0.10‒3.0
0.9997
0.1
‒
[39]
0.10‒70.00
0.9998
0.033
1.52
This work
Voltammetry Surface plasmon resonance assay Capillary
zone
electrophoresis
amperometric detection
sensor Liquid
chromatography-tandem
spectrometry Solid
phase
chromatography Fluorescence quenching based on BSAAuNCs 324 325
15
326
Scheme 1 Scheme of the synthetic strategy for BSA-AuNCs and the principle of CA
327
sensing.
328 329
330 331 332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355
Figure Captions Fig. 1. (A) Absorbance and fluorescence emission spectra of the BSA-AuNCs. Inset: photographic images under visible light (left) and ultraviolet light (right) of the BSAAuNCs. (B) Infrared spectra of (a) BSA and (b) BSA-AuNCs.
Fig. 2. Effect of (A) pH, (B) the amount of BSA-AuNCs, (C) reaction temperature and (D) reaction time on the fluorescence intensity of BSA-AuNCs-CAP system. F0 and F are the fluorescence intensity of BSA-AuNCs in the absence and presence of 2.50 µM CAP in phosphate buffer solution (30 mM, pH = 5.6).
Fig. 3. Effect of potentially interfering substances. CAP: 30.00 µΜ; glycine, histidine, L-cysteine, lysine, phenylalanine, aspartic acid, Na+, K+, Ca2+, and Mg2+: 6.00 mM and others: 3.00 mM. The inset displays the fluorescence spectra for potentially interfering substances. 50 µM AuNCs in phosphate buffer solution (30 mM, pH = 5.6) is used
Fig. 4. The change in fluorescence intensity of BSA-AuNCs upon the addition of various concentrations of CAP. The concentrations of CAP are: (a) 0.00, (b) 0.10, (c) 1.25, (d) 2.50, (e) 5.00, (f) 10.00, (g) 20.00, (h) 30.00, (i) 40.00, (j) 50.00, (k) 60.00, and (l) 70.00 µM. The inset displays the linear plot of F0/F against CCAP concentration of CAP. 50 µM AuNCs in phosphate buffer solution (30 mM, pH = 5.6) is used.
17
Fig. 1.
356 357 358 0.8 A
0.4 400
0.2 200
0.0 0
400
500
600
Wavelength (nm)
700
800
(b) BSA-AuNCs Transmittance (%)
600
Fluorescence Intensity (a.u.)
Absorbance (a.u.)
0.6
300
B
800
4000
(a) BSA
3500
3000
2500
2000
1500 -1 Wavenumber (cm )
1000
500
18
Fig. 2.
359
1.16
1.15
A
1.14
1.14
1.12
1.13
1.10
F0/F
F0/F
B
1.08
1.12 1.11
1.06 1.10
1.04
4
5
6
7
0.00
8
0.02
0.06
0.08
0.10
CBSA-AuNPs (mM)
pH
1.16
0.04
1.16
C
D
1.14
1.14
1.12 1.10 F0/F
F0/F
1.12 1.10
1.08 1.06
1.08
1.04
1.06
1.02
0
10
20
30
40
Temperature ( C) O
50
0
5
10
15
20
25
Time (min)
360 361
19
Fig. 3.
362
1.8 1.7 1.6
F0/F
1.5 1.4 1.3 1.2 1.1
Fluorescence intensity (a.u.)
500
Gly, His, Lys, Ser, Met, Val, Leu, Hcy, Cys, Phe, Asp, Na+, Zn2+, K+,
Blank
400
Ca2+, Mg2+ CAP
300 200 100 550
600
650
700
750
800
Wavelength (nm)
1.0
Bl
an k G ly H is Ly s Ph Ae sp Se Mr et V al Le u H cy C ys N + a K+ C 2 a + Zn 2 + M g 2+ C A P
BlankGly His Lys PheAsp Ser Met Val Leu Hcy CysNa+ K+Ca2+ Zn2+ Mg2+CAP
363
20
Fig. 4.
364
F0/F
Fluorescence intensity (a.u.)
2.8 F0/F=0.0253C+1.0719 R2=0.9996 2.4
a
600
500
2.0 1.6 1.2
400
0
10 20 30 40 50 60 70 80 CCAP (µM)
µM
l
300
200
100
0 550
600
650
700
Wavelength (nm) 365 366
750
800
367
Highlights
368
Bovine serum albumin stabilized Au nanoclusters were synthesized in aqueous solution.
369
The protein-stabilized water-soluble Au nanoclusters showed well fluorescence properties.
370
A novel fluorescent sensor for chloramphenicol was proposed.
371 372
22
373
Graphical abstract
374
23
