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A fluorescence detection of d -penicillamine based on Cu2+-induced fluorescence quenching system of protein-stabilized gold nanoclusters
Accepted Manuscript A fluorescence detection of D-penicillamine based on Cu2+-induced fluorescence quenching system of protein-stabilized gold nanoclusters Peng Wang, Bang Lin Li, Nian Bing Li, Hong Qun Luo PII: DOI: Reference:
S1386-1425(14)00970-6 http://dx.doi.org/10.1016/j.saa.2014.06.082 SAA 12341
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 January 2014 24 April 2014 1 June 2014
Please cite this article as: P. Wang, B.L. Li, N.B. Li, H.Q. Luo, A fluorescence detection of D-penicillamine based on Cu2+-induced fluorescence quenching system of protein-stabilized gold nanoclusters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.06.082
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.
A fluorescence
detection
of
Cu2+-induced
fluorescence
D-penicillamine quenching
based
system
on of
protein-stabilized gold nanoclusters Peng Wang, Bang Lin Li, Nian Bing Li, Hong Qun Luo Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
Corresponding author. Tel.: +86 23 68253237; Fax: +86 23 68253237. E-mail address: [email protected] (H.Q. Luo).
1
Abstract In this contribution, a luminescent gold nanoclusters which were synthesized by bovine serum albumin as novel fluorescent probes were successfully utilized for the determination of D-penicillamine for the first time. Cupric ion was employed to quench the strong fluorescence of the gold nanoclusters, whereas the addition of D-penicillamine caused obvious restoration of fluorescence intensity of the Cu2-gold nanoclusters system. Under optimum conditions, the increment in fluorescence intensity of Cu2-gold nanoclusters system caused by D-penicillamine was linearly proportional to the concentration of D-penicillamine in the range of 2.0 105 2.39 104 M. The detection limit for D-penicillamine was 5.4 106 M. With the off-on fluorescence signal at 650 nm approaching the near-infrared region, the present sensor for D-penicillamine detection had high sensitivity and low spectral interference. Furthermore, the novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine in real biological samples with satisfactory results.
Keywords: D-penicillamine; Fluorescence; Gold nanoclusters; Cupric ion; Turn-on sensor
2
1. Introduction Penicillamine (PA) is a naturally occurring sulfur-containing amino acid that belongs to the amino-thiols family. It is mainly derived from hydrolytic degradation of penicillin antibiotics [1]. Penicillamine can exist in D- and L-enantiomeric forms that show different biological and toxicological properties. However, only the D-enantiomer is clinically useful because of excessive toxicity of the L-type [2]. Thus, D-penicillamine (D-PA) is the drug of choice in the treatment of several pathologies, such as Wilson’s disease, rheumatoid arthritis, primary biliary cirrhosis, scleroderma, fibrotic lung diseases, cystinuria, and progressive systemic sclerosis. It is also an antidote in some heavy metal poisoning [3]. Currently, various analytical techniques have been reported for the determination of D-PA in both pharmaceutical preparations and biological samples. These methods include high performance liquid chromatography, chemiluminescence, flow injection analysis, capillary electrophoresis, electrochemistry, spectrophotometry, and fluorimetry [410]. Each method has its advantages and limitations, which would serve a particular need in analysis. Nevertheless, among the analytical methods mentioned above, the fluorescence-based assay has been widely used as a routine method for D-PA analysis due to its adequate sensitivity, convenience, low cost, simplicity, and easy operation. Recently, the development of suitable and fast procedures to determine D-PA by fluorimetry has attracted considerable interest. However, penicillamine is not detectable by direct spectroscopic techniques in biological fluids as it does not possess a chromophore. Therefore, derivatization of this compound is essential, prior to its determination. The presence of a carboxyl, thiol, and amino group in this compound provides a great deal of
3
ways
of
interaction
with
organic
and
inorganic
species
that
yield
some
spectrophotometrically active products [1,9,1113]. Nevertheless, these analysis methods of derivatization also suffer from some disadvantages, involving exploitation or utilization of environmental harmful dye, lack photostability, and a high spectral interference. So, it is highly desirable to develop simple, highly sensitive, and powerful fluorescent sensing systems for D-penicillamine. Recently, noble metal nanoparticles with fluorescence properties have attracted more and more attention due to their unique optical characteristics. They have been widely used in biosensors, biomarkers, biomedical imaging and so on [1419]. Among these fluorescent nanoparticles, one-pot protein-directed synthesis of Au nanoclusters (AuNCs) has high attraction for the simple synthetic process, and the resulting AuNCs with near-infrared emission are very stable over a wide range of pH conditions [20,21]. Near-infrared fluorescent probes have generated much interest in biosensor applications because biosensors operating in the near-infrared region can avoid interference from biological media and thereby facilitate relatively interference-free sensing [15,22]. With this protein-stabilized Au nanoclusters, various analytical methods have been developed for environmental, biochemical, and pharmaceutical analysis [14,15,23,24]. However, it has not been reported on the AuNCs as a novel, simple, and sensitive fluorescence probe for the determination of D-penicillamine. Herein, we synthesized gold nanoclusters with approaching near-infrared emission using bovine serum albumin (BSA) as a template according to the report [20]. With the addition of cupric ion, the fluorescence of the gold nanoclusters could be gradually quenched. While D-penicillamine was added subsequently, the fluorescence of the
4
analytical system was restored gradually. Based on the above phenomenon, an off-on switch fluorescence sensor for the determination of D-PA was developed with the advantages of simplicity, sensitivity, low-cost, and low spectral interference. With the present sensor, determination of trace amount of D-penicillamine was also realized in real biological samples, and satisfactory results were obtained.
2. Experimental 2.1. Apparatus A Hitachi F-4500 spectrofluorophotometer (Tokyo, Japan), which was equipped with a 150-W xenon lamp, was used for recording the fluorescence intensities and spectra with the slits (Ex/Em) of 5.0/5.0 nm and the PMT voltage of 700 V. A Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., Ltd., China) was used for recording the absorption spectra of the system. A pHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., Ltd., China) was used to measure the pH of the solution.
2.2. Reagents D-Penicillamine stock solution (0.0399 M) was prepared by dissolving 0.0595 g of D-penicillamine (99%, supplied by Sigma-Aldrich Co., USA) in doubly distilled water and diluting to the mark in a 10-mL calibrated cuvette. The working solution of D-penicillamine (8 mM) was obtained by diluting the stock solution with doubly distilled water. Cupric sulfate solution (1.0 mM) was prepared by dissolving 0.0254 g of copper sulfate pentahydrate (Chengdu Kelong Chemical Reagents Company, China) in doubly distilled water and diluting to the mark in a 100-mL calibrated flask. Bovine serum albumin
(BSA)
was
purchased
from
Sigma-Aldrich
Co.,
USA.
5
Tris(hydroxymethyl)aminomethane
(Tris)
and
4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from Aladdin Reagent Co., Shanghai, China. Phosphate buffer saline (PBS) (10 mM, pH 7.4), Britton-Robinson (BR) buffer (26 mM, pH 7.3), Na2HPO4NaH2PO4 (PB) buffer (100 mM, pH 7.4), Tris-HCl buffer (20 mM, pH 7.4), Tris-HAc buffer (25 mM, pH 7.4), Clark-Lubs buffer (52.7 mM, pH 7.4), and HEPES buffer (10 mM, pH 7.4) were prepared by the related reagents in proportion and pH values were adjusted using a pH meter. All the solutions noted above were stored at 0-4 C in a refrigerator. The BSA-stabilized gold nanoclusters (~0.8 nm) were synthesized in accordance with the method described in the previous reports [20,23]. In a typical experiment, all glassware used in the experiments were cleaned in a bath of freshly prepared aqua regia (HCl:HNO3 volume ratio 3:1), and rinsed thoroughly in doubly distilled water prior to use. Aqueous HAuCl4 solution (5 mL, 10 mM, 37 °C) was mixed with BSA solution (5 mL, 50 mg mL1, 37 °C) under vigorous stirring. Fresh NaOH solution (0.5 mL, 1 M) was introduced 2 min later, and the reaction was allowed to proceed under vigorous stirring at 37 C for 12 h. At last, the obtained product was stored at 4 C in a refrigerator when not in use. It can be kept for three months at least. All chemicals used in the experiments were of analytical reagent grade or the best grade commercially available and used as received without further purification. Doubly distilled water was used throughout the experiments.
2.3. Measurement procedure In a typical test, to 25 L of prepared gold nanoclusters solution in a 1.5 mL
6
eppendorf tube, 100 L of 100 mM phosphate buffer (pH 7.4) was added. Subsequently, 40 L of 1 mM cupric ion was added. Then appropriate volume of D-penicillamine working solution was added and the mixture was diluted to 1000 L with doubly distilled water. After shaking the solution and waiting for 5 min, the fluorescence spectra of the system were recorded in the region of 530 to 800 nm on excitation at 514 nm, and the fluorescence intensities were detected at 650 nm with the excitation and emission slit widths of 5.0 nm. All measurements were made at room temperature.
3. Results and discussion 3.1. Design strategy As shown in Fig. 1, the maximum emission peak of the gold nanoclusters was observed at 650 nm (curve 1), which displayed an approaching near-infrared emission upon excitation at 514 nm. The character of the spectra was consistent with the literature [14,25,26]. According to the previous studies, the approaching near-infrared emission was considered to arise from intra-band transitions of free electrons of the AuNCs. With the addition of cupric ion, the fluorescence of the gold nanoclusters could be quenched significantly (curve 2). The fluorescence quenching observed could be ascribed to the binding of cupric ion on to the bovine serum albumin (BSA) of gold nanoclusters through coordination [27]. Moreover, the paramagnetic nature of cupric ion can also facilitate quenching of the AuNCs fluorescence. The intersystem crossing of the excited electron from the BSA-stabilized gold nanoclusters was promoted in the presence of Cu2, which reduced the chance of electron transition from the excited state to the ground state with emission [28]. While D-penicillamine was added subsequently, the fluorescence of the
7
analytical system was restored (curve 3). This was because the cupric ion could also coordinate with the sulfhydryl group and carboxyl group of D-penicillamine and the coordination effect between D-PA and cupric ion was stronger than that between cupric ion and BSA of the AuNCs [24,28]. However, D-PA almost did not affect the fluorescence intensity of AuNCs (curve 4). The principle of the off-on fluorescence switch sensor in this contribution is depicted in Scheme 1. Hence, the turn-on fluorescence of the AuNCs-Cu2 could be used for the determination of D-penicillamine in a certain concentration range.
3.2. Optimization conditions for D-penicillamine analysis 3.2.1. Effect of the cupric ion concentration The quenching effect of cupric ion with different concentrations on the fluorescence intensity was observed after cupric ion was added to the gold nanoclusters solution. As shown in Fig. 2, with increasing concentrations of cupric ion in BSA-AuNCs solution, an obvious decrease in the fluorescence peak at 650 nm was clearly detected, and 40 M cupric ion quenched the fluorescence intensity largely and reached a plateau. However, excessive copper ion can also bind D-PA, which will decrease the effective concentration of D-PA and interfere with the determination. Therefore, taking the low probe background signals and wide linear range into account, 40 M cupric ion was chosen for subsequent studies. 3.2.2. Effect of the volume of gold nanoclusters In order to obtain a high sensitivity and wide linear range, the effect of volume of
8
AuNCs on the system was also examined. Generally, in the presence of quencher of a given concentration, the lower the concentration of fluorophore, the larger the change signal and thus the higher the sensitivity found. However, on the other hand, the signal-to-noise ratio (S/N) would be decreased when using too low concentration of fluorophore [29]. The results showed that when a large dose of AuNCs was used, the quenching effect of 40 M Cu2 was inconspicuous, leading to a low sensitivity for the D-PA detection. Contrarily, 40 M Cu2 could effectively quench fluorescence of small dosage of AuNCs with a favorable sensitivity and wide linear response for D-PA. Taking both sensitivity and linear range into consideration, 25 L portion of our prepared AuNCs was chosen for the following study. 3.2.3. Effect of the buffer Different kinds of buffers, including PBS, BR, PB, Tris-HCl, Tris-HAc, Clark-Lubs, and HEPES buffers, were used to investigate the effect of acidity on the relative fluorescence intensity of the AuNCs-Cu2-D-PA system. The results are shown in Fig. S1, from which it can be seen that different buffers had a little effect on the fluorescence of the analytical system, and the influence was so slight that it could be neglected. It has been reported that the AuNCs were directly utilized for biochemical analysis in double-distilled water [24]. Considering that the PB buffer provided the good performance for D-penicillamine detection in our work as shown in Fig. S1, and can be stored at room temperature and is relatively convenient to be used with low-cost, the PB buffer (pH 7.4) was used in this work. 3.2.4. Incubation time and stability of the system
9
The effects of time on the fluorescence intensities of AuNCs, AuNCs-Cu2, and AuNCs-Cu2-D-PA systems were also tested. The incubation time and stability were studied by determining the fluorescence intensities of the systems every 2 min for 2 h immediately after mixing. The results showed that the reaction between cupric ion and D-PA occurred rapidly at room temperature and the fluorescence intensity of the assay system reached the maximum in 5 min (Fig. S2). Moreover, the intensity remained constant for at least 30 min. Thus, this assay did not require crucial timing. 3.3. Calibration curve and detection limit Under optimum conditions, the fluorescence intensity of AuNCs-Cu2 increased with increasing concentration of D-PA, as shown in Fig. 3. Meanwhile, the maximum fluorescence wavelength was constant with increasing concentration of D-PA, indicating that the surface state of the BSA-AuNCs had no change. This phenomenon can further verify that it was the interaction between Cu2 and D-PA that caused the restoration of fluorescence intensity of the AuNCs-Cu2 system. Moreover, as shown in the inset of Fig. 3, there is a linear relationship between the relative fluorescence intensity (I = I – I0) and the concentration of D-PA over the range of 2.0 105 to 2.39 104 M, where I and I0 are the fluorescence intensity of the AuNCs-Cu2 system in the presence and absence of D-PA, respectively.
The linear regression equation can be expressed as I 1.036
1.996 c (c, 105 M) with correlation coefficient (R2) of 0.9910, where c is the concentration of D-PA. The detection limit is 5.4 μM for D-PA, which was given by 3σ/k, where σ is the standard deviation of the 11 blank determinations and k is the slope of the calibration curve. The repeatability of the proposed method was also evaluated by
10
performing a series of six repetitive experiments for the D-PA at the concentration of 80 µM, which provided a relative standard deviation (RSD) of 4.6%. This result suggests that our assay protocol is endowed with good repeatability. Comparisons with other methods for the determination of D-penicillamine are shown in Table 1. It can be seen that our approach has high sensitivity and will be a valuable tool for the determination of D-penicillamine.
3.4. Selectivity of the AuNCs-Cu2 system for detecting D-penicillamine In order to examine the selectivity of the AuNCs fluorescence sensor for D-PA determination, we investigated the fluorescence intensity of AuNCs-Cu2 system after addition of various other potential coexistent substances. The results are shown in Fig. 4 and from the figure we can see clearly that the fluorescence intensity of AuNCs-Cu2 system could be largely restored by the addition of D-PA. But with the addition of other substances such as maltose, sucrose, glucose, lactose, fructose, sodium gluconate, glutamic acid, lysine, glycine, valine, methionine, threonine, serine, alanine, asparagine, aspartic acid, isoleucine, phenylalanine, tyrosine, arginine, and tryptophan, the fluorescence of the analytical system changed little. The cysteine could enhance the fluorescence of the AuNCs-Cu2 analytical system a little, which was possibly considered that it was the coordination effect between cupric ion and cysteine that induced the enhancement of the fluorescence intensity [24]. While with the same concentration, the fluorescence of AuNCs-Cu2 system was much higher than that with the addition of cysteine. Thus, its influence could be neglected in the detection procedure. Therefore, we can conclude that the selectivity of the present assay was improved largely with a fluorescence switch procedure. Our approach has good sensitivity and selectivity. 11
3.5. Analytical application In order to validate the performance of the method for real samples analysis, the developed method was applied to the analysis of D-PA in human blood samples. The human blood samples were collected from healthy volunteers treated in the local hospital and was pretreated according to the previous report [37]. An aliquot of standard aqueous solution of D-penicillamine was added to 1 mL of blood sample, 1 mL of 0.60 M trichloroacetic acid solution was introduced and the mixture was shaken vigorously for 2 min to deposit proteins. The sample was blended on a vortex mixer and centrifuged at 6000 rpm/min for 20 min. The protein-free supernatant was transferred into 10 mL volumetric flask and the above procedure was then followed for recovery analysis. The results of real samples by standard addition method are summarized in Table 2. It can be seen that the recoveries of D-PA for individual samples varied between 100.8 and 107.5 . The relative standard deviations (RSD) for the three samples were between 1.5 % and 4.6 %. Therefore, our developed off-on fluorescence sensor can be applied for the determination of D-PA in real biological sample with satisfactory results.
4. Conclusions In this study, a novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine for the first time. In contrast to the previously reported detection methods for D-penicillamine, this new fluorescence assay exhibits high sensitivity, selectivity, and low spectral interference because of approaching near-infrared emission and off-on fluorescence sensing. The fluorescent sensor is simple, sensitive, and cost-effective. With the present sensor, determination of trace amount
12
D-penicillamine is realized in real biological samples with satisfactory results. The developed assay expands the application range of the gold nanoclusters synthesized by protein and develops the scientific idea of the off-on fluorescence sensing.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21273174 and 20975083) and the Municipal Science Foundation of Chongqing City (No. CSTC–2013jjB00002).
Appendix A. Supplementary material Supplementary data associated with this article (Figs. S1-S2) can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.xxxxx
13
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Figure and Table captions Scheme 1. The principle of the detection of D-PA. Fig. 1. Fluorescence spectra of AuNCs-Cu2-D-PA system. (1) AuNCs; (2) AuNCs-Cu2; (3) AuNCs-Cu2-D-PA; (4) AuNCs-D-PA. Conditions: AuNCs, 25 L; Cu2, 40 M; D-PA, 80 M; PB buffer, pH 7.4. Fig. 2. Fluorescence spectra of BSA-AuNCs in the presence of different concentrations of Cu2. The concentration of Cu2 (from 1 to 12, M): 0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0. Inset: The plot I/I0 versus the concentration of cupric ion. I0 and I are the fluorescence intensity of AuNCs in the absence and presence of Cu2+, respectively. AuNCs, 25 L; PB buffer, pH 7.4. Fig. 3. Fluorescence spectra of AuNCs-Cu2 in the presence of different concentrations of D-penicillamine. The concentrations of D-penicillamine (from 1 to 8, 105 M): 0, 2.0, 4.0, 8.0 12.0, 16.0, 20.0, and 23.9. Conditions: AuNCs, 25 L; Cu2, 40 M; PB buffer, pH 7.4. Inset: The calibration curve for D-penicillamine. Fig. 4. Selectivity of the sensing system for D-PA detection against other potentially interfering substances. Conditions: AuNCs, 25 L; Cu2, 40 M; D-PA, 239 M; others substances, 240 M; PB buffer, pH 7.4. Table 1. Comparison of sensitivities of this work with those of other methods for the determination of D-PA. Table 2. Results of the determination of D-penicillamine in human serum samples.
16
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Table 1
Comparison of sensitivities of this work with those of other methods for the determination of D-PA.
Method
Linear range (μM)
Detection limit (M)
Reference
Fluorimetry
33.5 536.2
0.7
[1]
Infrared spectroscopy
26.8 2546.7
3.4
[3]
Spectrophotometry
167.5 2010.6
Not given
[9]
Spectrophotometry
26.8 134.0
1.0
[30]
Electrochemical sensor
0.8 200.0
0.3
[31]
Electrochemical sensor
60.0 1600.0
46.0
[32]
Electrochemical sensor
65.0 – 1100.0
63.0
[33]
Electrochemical sensor
0.4 – 700
0.07
[34]
Electrochemical sensor
10.0 400.0
3.5
[35]
Electrochemical sensor
0.034 – 3.351
0.010
[36]
Fluorimetry
20.0 239.4
5.4
This work
Table 2
Results of the determination of D-penicillamine in human serum samples.
Added
Found a
Recovery
RSD
(105 M)
(105 M)
(%)
(%)
1
4.0
4.3
107.5
4.6
2
8.0
8.2
102.5
3.4
3
12.0
12.1
100.8
1.5
Sample
a
The mean of three experiments.
Highlights Gold nanoclusters as fluorescent probes were applied to detection of D-penicillamine. A novel and simple fluorescent sensor was established for D-penicillamine detection. The novel fluorescent assay exhibited high sensitivity and low spectral interference. This method was potentially useful in medicine assay in analytical chemistry.
Graphical Abstract
1
S1386-1425(14)00970-6 http://dx.doi.org/10.1016/j.saa.2014.06.082 SAA 12341
To appear in:
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy
Received Date: Revised Date: Accepted Date:
24 January 2014 24 April 2014 1 June 2014
Please cite this article as: P. Wang, B.L. Li, N.B. Li, H.Q. Luo, A fluorescence detection of D-penicillamine based on Cu2+-induced fluorescence quenching system of protein-stabilized gold nanoclusters, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2014), doi: http://dx.doi.org/10.1016/j.saa.2014.06.082
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.
A fluorescence
detection
of
Cu2+-induced
fluorescence
D-penicillamine quenching
based
system
on of
protein-stabilized gold nanoclusters Peng Wang, Bang Lin Li, Nian Bing Li, Hong Qun Luo Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China
Corresponding author. Tel.: +86 23 68253237; Fax: +86 23 68253237. E-mail address: [email protected] (H.Q. Luo).
1
Abstract In this contribution, a luminescent gold nanoclusters which were synthesized by bovine serum albumin as novel fluorescent probes were successfully utilized for the determination of D-penicillamine for the first time. Cupric ion was employed to quench the strong fluorescence of the gold nanoclusters, whereas the addition of D-penicillamine caused obvious restoration of fluorescence intensity of the Cu2-gold nanoclusters system. Under optimum conditions, the increment in fluorescence intensity of Cu2-gold nanoclusters system caused by D-penicillamine was linearly proportional to the concentration of D-penicillamine in the range of 2.0 105 2.39 104 M. The detection limit for D-penicillamine was 5.4 106 M. With the off-on fluorescence signal at 650 nm approaching the near-infrared region, the present sensor for D-penicillamine detection had high sensitivity and low spectral interference. Furthermore, the novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine in real biological samples with satisfactory results.
Keywords: D-penicillamine; Fluorescence; Gold nanoclusters; Cupric ion; Turn-on sensor
2
1. Introduction Penicillamine (PA) is a naturally occurring sulfur-containing amino acid that belongs to the amino-thiols family. It is mainly derived from hydrolytic degradation of penicillin antibiotics [1]. Penicillamine can exist in D- and L-enantiomeric forms that show different biological and toxicological properties. However, only the D-enantiomer is clinically useful because of excessive toxicity of the L-type [2]. Thus, D-penicillamine (D-PA) is the drug of choice in the treatment of several pathologies, such as Wilson’s disease, rheumatoid arthritis, primary biliary cirrhosis, scleroderma, fibrotic lung diseases, cystinuria, and progressive systemic sclerosis. It is also an antidote in some heavy metal poisoning [3]. Currently, various analytical techniques have been reported for the determination of D-PA in both pharmaceutical preparations and biological samples. These methods include high performance liquid chromatography, chemiluminescence, flow injection analysis, capillary electrophoresis, electrochemistry, spectrophotometry, and fluorimetry [410]. Each method has its advantages and limitations, which would serve a particular need in analysis. Nevertheless, among the analytical methods mentioned above, the fluorescence-based assay has been widely used as a routine method for D-PA analysis due to its adequate sensitivity, convenience, low cost, simplicity, and easy operation. Recently, the development of suitable and fast procedures to determine D-PA by fluorimetry has attracted considerable interest. However, penicillamine is not detectable by direct spectroscopic techniques in biological fluids as it does not possess a chromophore. Therefore, derivatization of this compound is essential, prior to its determination. The presence of a carboxyl, thiol, and amino group in this compound provides a great deal of
3
ways
of
interaction
with
organic
and
inorganic
species
that
yield
some
spectrophotometrically active products [1,9,1113]. Nevertheless, these analysis methods of derivatization also suffer from some disadvantages, involving exploitation or utilization of environmental harmful dye, lack photostability, and a high spectral interference. So, it is highly desirable to develop simple, highly sensitive, and powerful fluorescent sensing systems for D-penicillamine. Recently, noble metal nanoparticles with fluorescence properties have attracted more and more attention due to their unique optical characteristics. They have been widely used in biosensors, biomarkers, biomedical imaging and so on [1419]. Among these fluorescent nanoparticles, one-pot protein-directed synthesis of Au nanoclusters (AuNCs) has high attraction for the simple synthetic process, and the resulting AuNCs with near-infrared emission are very stable over a wide range of pH conditions [20,21]. Near-infrared fluorescent probes have generated much interest in biosensor applications because biosensors operating in the near-infrared region can avoid interference from biological media and thereby facilitate relatively interference-free sensing [15,22]. With this protein-stabilized Au nanoclusters, various analytical methods have been developed for environmental, biochemical, and pharmaceutical analysis [14,15,23,24]. However, it has not been reported on the AuNCs as a novel, simple, and sensitive fluorescence probe for the determination of D-penicillamine. Herein, we synthesized gold nanoclusters with approaching near-infrared emission using bovine serum albumin (BSA) as a template according to the report [20]. With the addition of cupric ion, the fluorescence of the gold nanoclusters could be gradually quenched. While D-penicillamine was added subsequently, the fluorescence of the
4
analytical system was restored gradually. Based on the above phenomenon, an off-on switch fluorescence sensor for the determination of D-PA was developed with the advantages of simplicity, sensitivity, low-cost, and low spectral interference. With the present sensor, determination of trace amount of D-penicillamine was also realized in real biological samples, and satisfactory results were obtained.
2. Experimental 2.1. Apparatus A Hitachi F-4500 spectrofluorophotometer (Tokyo, Japan), which was equipped with a 150-W xenon lamp, was used for recording the fluorescence intensities and spectra with the slits (Ex/Em) of 5.0/5.0 nm and the PMT voltage of 700 V. A Shimadzu UV-2450 spectrophotometer (Suzhou Shimadzu Instrument Co., Ltd., China) was used for recording the absorption spectra of the system. A pHS-3C pH meter (Shanghai Precision & Scientific Instrument Co., Ltd., China) was used to measure the pH of the solution.
2.2. Reagents D-Penicillamine stock solution (0.0399 M) was prepared by dissolving 0.0595 g of D-penicillamine (99%, supplied by Sigma-Aldrich Co., USA) in doubly distilled water and diluting to the mark in a 10-mL calibrated cuvette. The working solution of D-penicillamine (8 mM) was obtained by diluting the stock solution with doubly distilled water. Cupric sulfate solution (1.0 mM) was prepared by dissolving 0.0254 g of copper sulfate pentahydrate (Chengdu Kelong Chemical Reagents Company, China) in doubly distilled water and diluting to the mark in a 100-mL calibrated flask. Bovine serum albumin
(BSA)
was
purchased
from
Sigma-Aldrich
Co.,
USA.
5
Tris(hydroxymethyl)aminomethane
(Tris)
and
4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid (HEPES) were purchased from Aladdin Reagent Co., Shanghai, China. Phosphate buffer saline (PBS) (10 mM, pH 7.4), Britton-Robinson (BR) buffer (26 mM, pH 7.3), Na2HPO4NaH2PO4 (PB) buffer (100 mM, pH 7.4), Tris-HCl buffer (20 mM, pH 7.4), Tris-HAc buffer (25 mM, pH 7.4), Clark-Lubs buffer (52.7 mM, pH 7.4), and HEPES buffer (10 mM, pH 7.4) were prepared by the related reagents in proportion and pH values were adjusted using a pH meter. All the solutions noted above were stored at 0-4 C in a refrigerator. The BSA-stabilized gold nanoclusters (~0.8 nm) were synthesized in accordance with the method described in the previous reports [20,23]. In a typical experiment, all glassware used in the experiments were cleaned in a bath of freshly prepared aqua regia (HCl:HNO3 volume ratio 3:1), and rinsed thoroughly in doubly distilled water prior to use. Aqueous HAuCl4 solution (5 mL, 10 mM, 37 °C) was mixed with BSA solution (5 mL, 50 mg mL1, 37 °C) under vigorous stirring. Fresh NaOH solution (0.5 mL, 1 M) was introduced 2 min later, and the reaction was allowed to proceed under vigorous stirring at 37 C for 12 h. At last, the obtained product was stored at 4 C in a refrigerator when not in use. It can be kept for three months at least. All chemicals used in the experiments were of analytical reagent grade or the best grade commercially available and used as received without further purification. Doubly distilled water was used throughout the experiments.
2.3. Measurement procedure In a typical test, to 25 L of prepared gold nanoclusters solution in a 1.5 mL
6
eppendorf tube, 100 L of 100 mM phosphate buffer (pH 7.4) was added. Subsequently, 40 L of 1 mM cupric ion was added. Then appropriate volume of D-penicillamine working solution was added and the mixture was diluted to 1000 L with doubly distilled water. After shaking the solution and waiting for 5 min, the fluorescence spectra of the system were recorded in the region of 530 to 800 nm on excitation at 514 nm, and the fluorescence intensities were detected at 650 nm with the excitation and emission slit widths of 5.0 nm. All measurements were made at room temperature.
3. Results and discussion 3.1. Design strategy As shown in Fig. 1, the maximum emission peak of the gold nanoclusters was observed at 650 nm (curve 1), which displayed an approaching near-infrared emission upon excitation at 514 nm. The character of the spectra was consistent with the literature [14,25,26]. According to the previous studies, the approaching near-infrared emission was considered to arise from intra-band transitions of free electrons of the AuNCs. With the addition of cupric ion, the fluorescence of the gold nanoclusters could be quenched significantly (curve 2). The fluorescence quenching observed could be ascribed to the binding of cupric ion on to the bovine serum albumin (BSA) of gold nanoclusters through coordination [27]. Moreover, the paramagnetic nature of cupric ion can also facilitate quenching of the AuNCs fluorescence. The intersystem crossing of the excited electron from the BSA-stabilized gold nanoclusters was promoted in the presence of Cu2, which reduced the chance of electron transition from the excited state to the ground state with emission [28]. While D-penicillamine was added subsequently, the fluorescence of the
7
analytical system was restored (curve 3). This was because the cupric ion could also coordinate with the sulfhydryl group and carboxyl group of D-penicillamine and the coordination effect between D-PA and cupric ion was stronger than that between cupric ion and BSA of the AuNCs [24,28]. However, D-PA almost did not affect the fluorescence intensity of AuNCs (curve 4). The principle of the off-on fluorescence switch sensor in this contribution is depicted in Scheme 1. Hence, the turn-on fluorescence of the AuNCs-Cu2 could be used for the determination of D-penicillamine in a certain concentration range.
3.2. Optimization conditions for D-penicillamine analysis 3.2.1. Effect of the cupric ion concentration The quenching effect of cupric ion with different concentrations on the fluorescence intensity was observed after cupric ion was added to the gold nanoclusters solution. As shown in Fig. 2, with increasing concentrations of cupric ion in BSA-AuNCs solution, an obvious decrease in the fluorescence peak at 650 nm was clearly detected, and 40 M cupric ion quenched the fluorescence intensity largely and reached a plateau. However, excessive copper ion can also bind D-PA, which will decrease the effective concentration of D-PA and interfere with the determination. Therefore, taking the low probe background signals and wide linear range into account, 40 M cupric ion was chosen for subsequent studies. 3.2.2. Effect of the volume of gold nanoclusters In order to obtain a high sensitivity and wide linear range, the effect of volume of
8
AuNCs on the system was also examined. Generally, in the presence of quencher of a given concentration, the lower the concentration of fluorophore, the larger the change signal and thus the higher the sensitivity found. However, on the other hand, the signal-to-noise ratio (S/N) would be decreased when using too low concentration of fluorophore [29]. The results showed that when a large dose of AuNCs was used, the quenching effect of 40 M Cu2 was inconspicuous, leading to a low sensitivity for the D-PA detection. Contrarily, 40 M Cu2 could effectively quench fluorescence of small dosage of AuNCs with a favorable sensitivity and wide linear response for D-PA. Taking both sensitivity and linear range into consideration, 25 L portion of our prepared AuNCs was chosen for the following study. 3.2.3. Effect of the buffer Different kinds of buffers, including PBS, BR, PB, Tris-HCl, Tris-HAc, Clark-Lubs, and HEPES buffers, were used to investigate the effect of acidity on the relative fluorescence intensity of the AuNCs-Cu2-D-PA system. The results are shown in Fig. S1, from which it can be seen that different buffers had a little effect on the fluorescence of the analytical system, and the influence was so slight that it could be neglected. It has been reported that the AuNCs were directly utilized for biochemical analysis in double-distilled water [24]. Considering that the PB buffer provided the good performance for D-penicillamine detection in our work as shown in Fig. S1, and can be stored at room temperature and is relatively convenient to be used with low-cost, the PB buffer (pH 7.4) was used in this work. 3.2.4. Incubation time and stability of the system
9
The effects of time on the fluorescence intensities of AuNCs, AuNCs-Cu2, and AuNCs-Cu2-D-PA systems were also tested. The incubation time and stability were studied by determining the fluorescence intensities of the systems every 2 min for 2 h immediately after mixing. The results showed that the reaction between cupric ion and D-PA occurred rapidly at room temperature and the fluorescence intensity of the assay system reached the maximum in 5 min (Fig. S2). Moreover, the intensity remained constant for at least 30 min. Thus, this assay did not require crucial timing. 3.3. Calibration curve and detection limit Under optimum conditions, the fluorescence intensity of AuNCs-Cu2 increased with increasing concentration of D-PA, as shown in Fig. 3. Meanwhile, the maximum fluorescence wavelength was constant with increasing concentration of D-PA, indicating that the surface state of the BSA-AuNCs had no change. This phenomenon can further verify that it was the interaction between Cu2 and D-PA that caused the restoration of fluorescence intensity of the AuNCs-Cu2 system. Moreover, as shown in the inset of Fig. 3, there is a linear relationship between the relative fluorescence intensity (I = I – I0) and the concentration of D-PA over the range of 2.0 105 to 2.39 104 M, where I and I0 are the fluorescence intensity of the AuNCs-Cu2 system in the presence and absence of D-PA, respectively.
The linear regression equation can be expressed as I 1.036
1.996 c (c, 105 M) with correlation coefficient (R2) of 0.9910, where c is the concentration of D-PA. The detection limit is 5.4 μM for D-PA, which was given by 3σ/k, where σ is the standard deviation of the 11 blank determinations and k is the slope of the calibration curve. The repeatability of the proposed method was also evaluated by
10
performing a series of six repetitive experiments for the D-PA at the concentration of 80 µM, which provided a relative standard deviation (RSD) of 4.6%. This result suggests that our assay protocol is endowed with good repeatability. Comparisons with other methods for the determination of D-penicillamine are shown in Table 1. It can be seen that our approach has high sensitivity and will be a valuable tool for the determination of D-penicillamine.
3.4. Selectivity of the AuNCs-Cu2 system for detecting D-penicillamine In order to examine the selectivity of the AuNCs fluorescence sensor for D-PA determination, we investigated the fluorescence intensity of AuNCs-Cu2 system after addition of various other potential coexistent substances. The results are shown in Fig. 4 and from the figure we can see clearly that the fluorescence intensity of AuNCs-Cu2 system could be largely restored by the addition of D-PA. But with the addition of other substances such as maltose, sucrose, glucose, lactose, fructose, sodium gluconate, glutamic acid, lysine, glycine, valine, methionine, threonine, serine, alanine, asparagine, aspartic acid, isoleucine, phenylalanine, tyrosine, arginine, and tryptophan, the fluorescence of the analytical system changed little. The cysteine could enhance the fluorescence of the AuNCs-Cu2 analytical system a little, which was possibly considered that it was the coordination effect between cupric ion and cysteine that induced the enhancement of the fluorescence intensity [24]. While with the same concentration, the fluorescence of AuNCs-Cu2 system was much higher than that with the addition of cysteine. Thus, its influence could be neglected in the detection procedure. Therefore, we can conclude that the selectivity of the present assay was improved largely with a fluorescence switch procedure. Our approach has good sensitivity and selectivity. 11
3.5. Analytical application In order to validate the performance of the method for real samples analysis, the developed method was applied to the analysis of D-PA in human blood samples. The human blood samples were collected from healthy volunteers treated in the local hospital and was pretreated according to the previous report [37]. An aliquot of standard aqueous solution of D-penicillamine was added to 1 mL of blood sample, 1 mL of 0.60 M trichloroacetic acid solution was introduced and the mixture was shaken vigorously for 2 min to deposit proteins. The sample was blended on a vortex mixer and centrifuged at 6000 rpm/min for 20 min. The protein-free supernatant was transferred into 10 mL volumetric flask and the above procedure was then followed for recovery analysis. The results of real samples by standard addition method are summarized in Table 2. It can be seen that the recoveries of D-PA for individual samples varied between 100.8 and 107.5 . The relative standard deviations (RSD) for the three samples were between 1.5 % and 4.6 %. Therefore, our developed off-on fluorescence sensor can be applied for the determination of D-PA in real biological sample with satisfactory results.
4. Conclusions In this study, a novel gold nanoclusters-based fluorescent sensor has been applied to the determination of D-penicillamine for the first time. In contrast to the previously reported detection methods for D-penicillamine, this new fluorescence assay exhibits high sensitivity, selectivity, and low spectral interference because of approaching near-infrared emission and off-on fluorescence sensing. The fluorescent sensor is simple, sensitive, and cost-effective. With the present sensor, determination of trace amount
12
D-penicillamine is realized in real biological samples with satisfactory results. The developed assay expands the application range of the gold nanoclusters synthesized by protein and develops the scientific idea of the off-on fluorescence sensing.
Acknowledgements This work was supported by the National Natural Science Foundation of China (Nos. 21273174 and 20975083) and the Municipal Science Foundation of Chongqing City (No. CSTC–2013jjB00002).
Appendix A. Supplementary material Supplementary data associated with this article (Figs. S1-S2) can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.xxxxx
13
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Figure and Table captions Scheme 1. The principle of the detection of D-PA. Fig. 1. Fluorescence spectra of AuNCs-Cu2-D-PA system. (1) AuNCs; (2) AuNCs-Cu2; (3) AuNCs-Cu2-D-PA; (4) AuNCs-D-PA. Conditions: AuNCs, 25 L; Cu2, 40 M; D-PA, 80 M; PB buffer, pH 7.4. Fig. 2. Fluorescence spectra of BSA-AuNCs in the presence of different concentrations of Cu2. The concentration of Cu2 (from 1 to 12, M): 0, 5.0, 10.0, 15.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0, 55.0. Inset: The plot I/I0 versus the concentration of cupric ion. I0 and I are the fluorescence intensity of AuNCs in the absence and presence of Cu2+, respectively. AuNCs, 25 L; PB buffer, pH 7.4. Fig. 3. Fluorescence spectra of AuNCs-Cu2 in the presence of different concentrations of D-penicillamine. The concentrations of D-penicillamine (from 1 to 8, 105 M): 0, 2.0, 4.0, 8.0 12.0, 16.0, 20.0, and 23.9. Conditions: AuNCs, 25 L; Cu2, 40 M; PB buffer, pH 7.4. Inset: The calibration curve for D-penicillamine. Fig. 4. Selectivity of the sensing system for D-PA detection against other potentially interfering substances. Conditions: AuNCs, 25 L; Cu2, 40 M; D-PA, 239 M; others substances, 240 M; PB buffer, pH 7.4. Table 1. Comparison of sensitivities of this work with those of other methods for the determination of D-PA. Table 2. Results of the determination of D-penicillamine in human serum samples.
16
Scheme 1
Figure 1
Figure 2
Figure 3
Figure 4
Table 1
Comparison of sensitivities of this work with those of other methods for the determination of D-PA.
Method
Linear range (μM)
Detection limit (M)
Reference
Fluorimetry
33.5 536.2
0.7
[1]
Infrared spectroscopy
26.8 2546.7
3.4
[3]
Spectrophotometry
167.5 2010.6
Not given
[9]
Spectrophotometry
26.8 134.0
1.0
[30]
Electrochemical sensor
0.8 200.0
0.3
[31]
Electrochemical sensor
60.0 1600.0
46.0
[32]
Electrochemical sensor
65.0 – 1100.0
63.0
[33]
Electrochemical sensor
0.4 – 700
0.07
[34]
Electrochemical sensor
10.0 400.0
3.5
[35]
Electrochemical sensor
0.034 – 3.351
0.010
[36]
Fluorimetry
20.0 239.4
5.4
This work
Table 2
Results of the determination of D-penicillamine in human serum samples.
Added
Found a
Recovery
RSD
(105 M)
(105 M)
(%)
(%)
1
4.0
4.3
107.5
4.6
2
8.0
8.2
102.5
3.4
3
12.0
12.1
100.8
1.5
Sample
a
The mean of three experiments.
Highlights Gold nanoclusters as fluorescent probes were applied to detection of D-penicillamine. A novel and simple fluorescent sensor was established for D-penicillamine detection. The novel fluorescent assay exhibited high sensitivity and low spectral interference. This method was potentially useful in medicine assay in analytical chemistry.
Graphical Abstract
1