Author’s Accepted Manuscript Gold-nanoparticle-based colorimetric array for detection of dopamine in urine and serum Yumin Leng, Kun Xie, Liqun Ye, Genquan Li, Zhiwen Lu, Junbao He www.elsevier.com
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To appear in: Talanta Received date: 30 December 2014 Revised date: 17 February 2015 Accepted date: 20 February 2015 Cite this article as: Yumin Leng, Kun Xie, Liqun Ye, Genquan Li, Zhiwen Lu and Junbao He, Gold-nanoparticle-based colorimetric array for detection of dopamine in urine and serum, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.038 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 galley proof before it is published in its final citable 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.
Gold-nanoparticle-based colorimetric array for detection of dopamine in urine and serum Yumin Leng1*, Kun Xie1, Liqun Ye2, Genquan Li1, Zhiwen Lu1, Junbao He1 1
College of Physics and Electronic Engineering, Nanyang Normal University, Nanyang 473061,
China. 2
College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang
473061, China.
Corresponding Author *E-mail: [email protected] ABSTRACT: A highly selective method is presented for the colorimetric determination of dopamine (DA) using gold nanoparticles (AuNPs). DA caps on the surface of AuNPs (DAAuNPs) and induces the aggregation of AuNPs in alkaline solution. The DA-AuNPs are modified by the hydrolysate of thioglycolic acid (TGA2-) through Au-S bonds. The aggregation of AuNPs is accelerated by TGA2-, due to the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA. Upon the addition of DA, the solution shows a color change from red to purple (or yellow), which is also monitored to detect DA in human urine and fetal bovine serum samples. Here, the limits of colorimetric detection are as low as 10-7 M observed in Milli-Q water, urine and serum. Based on UV-vis absorption spectra, the limits of detection have been calculated to be 3.310-8 M, 1.010-7 M and 9.410-8 M in Milli-Q water, urine and serum, respectively. All the limits of detection are lower than the lowest abnormal concentrations of DA in urine (5.710-7 M) and blood (1.610-5 M). The good linear ranges from 0 to 10-6 M are used for the quantitative assay of DA in urine and serum samples. The applicability of our detection system is also verified by analysis of DA in urine and serum samples. The developed approach is without using complex financial instruments.
Keywords: Gold nanopartilces; colorimetric detection; dopamine; urine; serum 1. Introduction Dopamine (DA), a catecholamine derived from tyrosine, is an important neurotransmitter of the central and peripheral nervous systems [1,2]. It is widely distributed in the central neural system, brain tissues and body fluids of mammals and plays a vital role in the function of the central nervous, hormonal, renal and cardiovascular systems [3-6]. Normal level of DA in body supports blood pressure, fine motor activity, inspiration, intuition and focus, whereas abnormal DA concentrations in body often causes euphoria, addiction, obesity, even serious diseases such as schizophrenia, Parkinsonism, Alzheimer’s disease, Huntington’s disease, epilepsy, pheochromocytoma and neuroblastoma [3-10]. Moreover, hard drugs (sush as cocaine, opium, heroin, morphine, and codeine), doping, tobacco and alcohol can activate the secretion of DA in human body [11-15]. So it is necessary to determine the level of DA in human body fluid rapidly. To detect DA accurately, a number of physico-chemical methods such as fluorescence [16-18], electrochemical detection [19-21], and high performance liquid chromatography [22] have been reported. Although these methods are accurate, they are usually time-consuming, need complicated instruments or lack sensitivity. Thus, it still remains a great challenge to develop an inexpensive, fast and reliable method for determination of trace amount of DA in body fluid, such as blood and urine. Without the aid of expensive instruments, colorimetric method is convenient and can be easily monitored by the naked eyes [23-37]. Because of the unique distance-dependent optical property of AuNPs/silver nanoparticles (AgNPs), Au/AgNPs-based colorimeric assays have been widely used to detect analytes, such as ions, molecules and cell [27-37]. Au/AgNPsbased colorimeric methods for the detection of DA have been developed only recently [38-42]. For example, Qu group used the probe of AgNPs to detect DA in Milli-Q water [40]. To detect cerebral DA, Tian et al. designed AuNPs functionalized with two ligands [41]. Wang et
al. developed the functional AuNPs to determinate DA in human serum [42]. Although conceptually simple, the developed methods are usually limited by the low selectivity and the application in real systems. Therefore, substantial efforts are still needed for the development of efficient methods to meet the increasing demand for real DA detection with high selectivity. Herein, we develop a rapid, sensitive and selective approach for the colorimetric detection of DA using AuNPs, with theoretical simplicity and low technical demands. In alkaline solution, DA capps and aggregates AuNPs (DA-AuNPs) due to the covalent and noncovalent interactions between DA and AuNPs [43,44]. The DA-AuNPs are modified by the hydrolysate of thioglycolic acid (TGA2-) through Au-S bonds [30,34]. Due to the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA, the AuNPs are further aggregated [45,46]. With the increase of DA concentrations, the color of AuNPs gradually changes from an initial red to purple, then gray and finally yellow. The color changes are also monitored to detect DA in human urine and fetal bovine serum samples. The limits of detection (LOD) by the naked eyes are as low as 10-7 M in Milli-Q water and samples. The LODs by UV-vis spectroscopy are 3.310-8 M, 1.010-7 M and 9.410-8 M in Milli-Q water, urine and serum, respectively. All the LODs are lower than the lowest abnormal concentrations of DA and in urine (5.710-7 M) and blood (1.610-5 M) [47-49]. Moreover, the developed probe shows low response to 22 probable interferences comparing with DA. The interfering substances are considered comprehensively, comparising with those in the literature [38-42]. Therefore, the present method shows high selectivity for DA detection, and has been successfully applied for the detection of DA in urine and serum samples. 2. Experimental section 2.1 Materials and Instrumentation Chloroauric acid tetrahydrate (HAuCl4•4H2O), hexadecyl trimethyl ammonium bromide (CTAB), sodium borohydride (NaBH4), NaCl, KCl, Na2CO3, CaCl2, ZnCl2, NH4Cl, lactate,
uric acid, urea, oxalate, histidine, lysine, urea, and ascorbic acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Thioglycolic acid (TGA), FeCl2•4H2O, L-tyrosine, L-aspartic acid, threonine, glucose, dopamine hydrochloride, glycine, folic acid, ferulic acid and tryptophan were obtained from Aladdin-regent Co. Ltd (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, USA). All chemicals were used as received without further purification. Millipore-Q water was used throughout the experiment. All glassware were washed with aqua regia (HCl/HNO3 = 3:1 (v/v)) and then cleaned with Milli-Q water. Transmission electron microscopy (TEM) was performed on a Tencai F20 instrument with an acceleration voltage of 200 kV. UV-vis absorption spectra were recorded using a Lambda 950 UV-vis spectrophotometer from Perkin Elmer. 2.2 Preparation of AuNPs AuNPs were prepared through the reduction of HAuCl4 by NaBH4 in the presence of CTAB, according to Murphy's method [50]. In brief, 5 mL of 5 mM HAuCl4 solution was added into 95 mL double distilled water with vigorous stirring. 1 mL of 20 mM CTAB was injected into the mixture under vigorous stirring. Then, 1.5 mL of 0.1 M freshly prepared NaBH4 solution was added slowly during stirring. The reaction was kept for 30 min, during the time the color changed from pink to deep red. The prepared AuNPs solution was stored at 4 0C until use. 2.3 Colorimetric Detection of DA 2.3.1 Sensitivity To evaluate the detectable minimum concentration of DA, the typical colorimetric analysis was carried out by following steps. Firstly, 30 μL of DA solutions with different concentrations were mixed with 1380 μL AuNPs. Next, 30 μL NaOH (5 M) was added to the above mixture, 10 min later, 60 μL of 1% TGA was added, and the mixture was allowed to
react for 20 min at 25 0C. Color change was observed with the naked eye and UV-vis absorption spectra were recorded with a UV-vis spectrophotometer. 2.3.2 Selectivity The selectivity for DA was investigated as follows: 30 μL of 5×10-5 M the representative biomolecules and ions that probably exist in urine and serum, such as L-tyrosine (L-Tyr), lactate (Lac), uric acid (UA), L-aspartic acid (L-Asp), oxalate (Oxa), histidine (Hist), lysine (Lys), threonine (Thr), glucose (Gluc), glycine (Gly), folic acid (Foli), ferulic acid (Fer), urea, acrobatic acid (AA), L-tryptophan (L-Try), DA, Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-, were added into 1380 μL AuNPs, respectively. Next, 30 μL NaOH (5 M) was added into the above mixture, 10 min later, 60 μL of 1% TGA was added, and the mixture was allowed to react for 20 min at 25 0C. The final concentrations of analytes are 10-6 M. The corresponding photo images and UV-vis absorption spectra were recorded. 2.3.3 Practical application In order to assess the validity of the developed procedure, the method was applied to detect DA in biological fluids such as human urine and FBS samples. The spiked samples were obtained by adding DA with different concentration to the dialysates of human urine and FBS. The detection procedure of DA in biological fluids is similar with that in double distilled water. The corresponding photo images and UV-vis absorption spectra were recorded. 3. Results and discussion 3.1. Principle of DA Detection Scheme 1 shows the probable mechanism for the detection of DA. Due to the strong covalent and noncovalent interactions between DA and AuNPs [43,44], DA caps and aggregates AuNPs (DA-AuNPs) in alkaline solution (See Scheme 1B). The DA-AuNPs are modified by TGA2- ([SCH2CO2]2-) through Au-S bonds [30,34]. Due to the strong hydrogenbonds (NH···OC and OH···OC) formed between TGA2- and DA [45,46], the AuNPs are further aggregated (See Scheme 1C). Although the exact function of alkaline is unknown at
this time, it is likely to provide strong oxidizing environment, hence speeds up the reaction in the system. Moreover, under oxidizing conditions, DA reacts with thiols via Schiff base reaction [51]. Scheme 1 TEM characterizations are used to elucidate the principle of DA detection. TEM images (See Fig. S1) show the microstructures of the AuNPs in the absence and presence of 10-6 M DA and TGA2-. Notably, without DA, AuNPs are well monodispersed (See Fig. S1, panel A). Nevertheless, in the presence of 10-6 M DA, AuNPs start to aggregate due to the covalent and noncovalent interactions between DA and AuNPs [43], and DA capps on the surface of AuNPs (DA-AuNPs) in alkaline solution [44]. The aggregation of DA-AuNPs is accelerated by the inducer of TGA2- (See Fig. S1, panel C), because of the strong hydrogen-bond interactions between TGA2- and DA [45,46]. Meanwhile, DLS experimental results show that the sizes of AuNPs are also increased with the increase of DA concentration (See Fig. 1). Fig. 1 3.2 Optimization of the Expremental Parameters The performance of the as-developed method for DA detection may be strongly influenced by the experimental conditions such as pH of the colloidal solution, reaction temperature, and reaction time. Thus, each detection parameter was optimized in our study, while keeping other parameters constant. The effect of pH on the colorimetric detection of DA was investigated by adding different concentrations of NaOH, while keeping DA concentration unchanged (10-6 M). Comparing with the solution without DA (blank), the color change of the solution with 10-6 M DA can be immediately observed when the concentration of NaOH is beyond 5 M (See Fig. S2). At this time, the pH of the solution was measured to be 13, which was chosen in the following experiments.
As expected, the reaction temperature has great effect on DA detection. The UV-vis absorption spectra of the developed reagent in the absence and presence of 10-5 M DA, and the corresponding absorption ratio (Ablank/A[DA]=10-5 M) at different temperature are shown in Fig. 2. As shown in Fig. 2A, the UV-vis absorption peak of the developed reagent is redshifted when the reaction temperature is above 25 0C, indicating that AuNPs are unstable and aggregated at higher temperature (> 25 0C). Moreover, with the increase of temperature, the absorption ratio (Ablank/A[DA]=10-5 M) increases quickly before 25 0C, and slowly when the reaction temperature is above 25 0C (See Fig. 2, panel C). Thus, 25 0C was chosen as the optimal reaction temperature in the experiments. Fig. 2 The incubation time also influences the colorimetric response of the developed reagent in the presence of DA. To investigate the effect of reaction time, the changes of the UV-vis absorption spectra of the developed reagent with 10-6 M DA were recorded within 35 min and the absorption ratio of the minimum and maximum (Amin/Amax) were obtained, as shown in Fig. 3. The value of Amin/Amax increases exponentially with the increase of reaction time (See Fig. 3, panel B), and reaches an equilibrium almost after 20 min. Therefore, 20 min was chosen for the optimal incubation time in the experiments. Fig. 3 3.3 Colorimetric Detection of DA Using the Developed Method 3.3.1 Sensitivity In the absence of TGA2-, the limit of colorimetric detection is approximately 510-6 M (See Fig. 4, panel A), which is higher than the lowest abnormal concentration (5.710-7 M) of DA in urine [47]. In the presence of TGA2- and upon addition of increasing concentrations of DA, the color of AuNPs gradually changes from initially red to purple, then gray and finally yellow. And the limit of colorimetric detection is significantly proved to be 10-7 M (See Fig. 4, panel B), which is lower than the previously reported data (1.85×10-7 M) [41] (See Table S1
in the Supporting Information). This observation reveals that the sensitivity for DA detection can be greatly imporved by adding TGA2-. These results are further confirmed by UV-vis spectroscopy. The UV-vis absorption spectra of the developed reagent with different concentrations of DA are shown in Fig. 4, panel C. The absorption peak is clearly red-shifted and the absorption intensity decreases with the increase of DA concentration. The ratio (A0/A) of the absorption intensity (A0 and A are corresponding to the developed reagent with concentrations of DA (0 and 10-9 ~ 10-5 M).), was used to express the molar ratio between dispersed and aggregated AuNPs. The ratio (A0/A) as a function of DA concentration is shown in Fig. 4C inset. The linear relationship (R2=0.998) can be inferred, manifesting the dynamic range from 0 to 10-6 M and the LOD of 3.3×10-8 M (See the calculation method in the Supporting Information). As shown in Table S1 (see the Supporting Information), the linear range of this approach is wider than those of other AuNPs as colorimetric probes [41, 42]. The LOD of 3.3×10-8 M is lower than the reported data (5×10-8 M and 7×10-8 M) in the literature [41,42]. Moreover, the detection limits of the colorimetric assay and calculation for DA (10-7 M and 3.3×10-8 M) in Milli-Q water are lower than the lowest abnormal concentrations of DA (5.710-7 M and 1.610-5 M) in urine and blood [47-49]. Thus, the present strategy developed in this study is sensitive and able to realize the colorimetric detection of DA in real samples, such as urine and blood. Fig. 4 3.3.2 Selectivity The complexity of urine and serum presents a great challenge to the analytical methods for DA detection not only in the detection limit and sensitivity but, more importantly, in selectivity. The selective recognition of DA by the developed method was investigated by considering other 22 biomolecules and ions that probably exist in urine and serum, such as LTyr, Lac, UA, L-Asp, Oxa, Hist, Lys, Thr, Gluc, Gly, Foli, Fer, urea, AA, L-Try, Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-. The interfering substances are considered comprehensively,
comparising with those in the literature [38-42]. The concentrations of analytes are 10-6 M. The corresponding photo image and UV-vis absorption ratio (A580/A525) are shown in Fig. 5. It is clear that the developed probe has very high specificity toward DA (See Fig. 5). Fig. 5 3.3.3 Practical application As demonstrated above, the present colorimetric method for DA detection with high selectivity provides a direct platform for assaying DA in urine and serum. Thus, the developed method was applied in detection of DA in biological fluids such as human urine and FBS samples. The spiked samples were obtained by adding concentrations of DA to the dialysates of human urine and FBS. The corresponding photo images and UV-vis absorption spectra are shown in Fig. 6 and Fig. 7, respectively. The color of the solutions changes from red to purple, gray and yellow with the increase of DA concentrations (See Fig. 6 and Fig. 7, panels A). Although the complex elements in the spiked samples slightly affect the color changes (compared with Fig. 4, panel B), the limits of colorimetric detection are both up to 10-7 M. UV-vis spectroscopy provides a more precise approach for the detection of DA contained in the dialysates of human urine and FBS (See Fig. 6 and Fig. 7, panels B). As the contained concentrations of DA increase, the absorption peak is red-shifted and the absorption intensity decreases. The ratio (A0/A) as a function of DA concentration are shown in Fig. 6 and Fig. 7 B insets, respectively. The linear relationships can be inferred, manifesting the dynamic ranges from 0 to 10-6 M and the LODs of 1.0×10-7 M and 9.4×10-8 M (See the calculation method in the Supporting Information), respectively. The detection limits of the colorimetric assays (both 10-7 M) and calculations (1.0×10-7 M and 9.4×10-8 M) for DA in the dialysates of human urine and FBS are lower than the lowest abnormal DA concentrations of 5.710-7 M (urine) and 1.610-5 M (blood) [47-49], respectively. As shown in Table S1 (see the Supporting Information), we first present the analytical parameters of linearity and sensitivity in real application, comparing with those of other AuNPs as colorimetric probes
[41,42]. Moreover, the accuracy and precision of the developed procedure were assessed, as shown in Table 1. The found DA concentrations using the developed method are in good agreement with the spiked amount. The recoveries are within the range from 101.3 to 108.5%. Thus, the developed method has been successfully applied for the detection of DA in urine and serum samples. 4. Conclusions In summary, we have developed a rapid, sensitive, and highly selective strategy for the colorimetric visualization of DA. Because of the strong covalent and noncovalent interactions between DA and AuNPs, and the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA, so we observed that DA capped and aggregated AuNPs, and the aggregation was accelerated by TGA2-. The selectivity of the DA detection system by the naked eyes and UV-vis absorption spectra is excellent comparing with 22 probable interferences. The interfering substances are considered comprehensively, and we first present the analytical parameters of linearity and sensivity in real application, comparising with those in the literature. The limits of colorimetric detection have been determined to be 10-7 M in Milli-Q water, urine and serum. The LODs have been calculated to be 3.310-8 M (Milli-Q water), 1.0×10-7 M (urine) and 9.4×10-8 M (serum), respectively. All the LODs are lower than the lowest abnormal DA concentrations in urine (5.710-7 M) and blood (1.610-5 M). The good linear relationship between A0/A and DA concentration from 0 to 10-6 M, which are used for the quantitative assay of DA in urine and serum samples. These results indicate that the developed detection system is applicable for rapid colorimetric detection of DA with excellent selectivity and high sensitivity in urine and serum samples. Acknowledgements This work was supported by the Science and Technology Program of Education Department of Henan Province (No. 14A430024), Natural Science Foundation of Nanyang Normal University (Nos. zx2014087), National Natural Science Fund (E041604/51374132).
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C
B
A
H O
H O
H O
H O S
HNH
HNH O
TGA2-
DA
H O
O
H O
H O
O
H O O S
HNH HNH
DA
HO
H O
S
TGA2-
O
O
HNH
Scheme 1. Colorimetric detection of dopamine (DA) based on AuNPs: (A) disperse AuNPs, (B) AuNPs aggregated and capped by DA (DA-AuNPs), (C) AuNPs further aggregated by TGA2- ([SCH2CO2]2-).
A
20
[DA]/0M
Number (%)
Number (%)
25 20 15 10 5 0
[DA]/10 M
15 10 5 0
0
500
1000 1500 2000
0
500
Size (d.nm) [DA]/10 M
20 15 10 5 0 0
25
-6
C
500
1000 1500 2000
Size (d.nm)
1000 1500 2000
Number (%)
25
Number (%)
-7
B
-2
[DA]/10 M
D
20 15 10 5 0 0
500
Size (d.nm)
1000 1500 2000
Size (d.nm)
Fig. 1. Size distribution of the AuNPs measured by DLS in the (A) absence and presence of (B) 10-7 M, (C) 10-6 M, and (D) 10-2 M DA induced by TGA2-.
Absorbance (a. u.)
0.8
0
Temp. ( C)
A
0 10 15 20 25 30 35 40
0.6 0.4 0.2 400
500
600
700
800
Wavelength (nm)
Absorbance (a. u.)
0.7
0
Temp. ( C)
B
0 10 15 20 25 30 35 40
0.6 0.5 0.4 0.3 0.2 400
500
600
700
800
Wavelength (nm) 40
C
Temp. (0C)
30 20 10 0 1.0
1.2
1.4
1.6
1.8
2.0
2.2
Ablank/A[DA]=10-5M
Fig. 2. UV-vis absorption spectra of the developed reagent at different temperature in the (A) absence and (B) presence of 10-5 M DA. (C) Temperature-absorbance ratio of Ablank/A[DA]=10-5 M.
Absorbance (a. u.)
Time (min)
A
0.7
0 1 3 6 9 12 15 18 21 24 27 30 35
0.6 0.5 0.4 0.3 0.2 0.1 400
500
600
700
800
900
Wavelength (nm) 0.70
B
Amin/Amax
0.69 0.68 0.67 0.66 0.65 -5
0
5
10
15
20
25
30
35
40
Time (min) Fig. 3. (A) Time-dependent absorption spectra for the developed reagent containing 10-6 M DA. (B) Time-absorbance ratio of Amin/Amax.
Blank 10-9
10-8
10-7
10-6
10-4
5×10-6 10-5
10-3
10-2
A
B
10
0.5
10-4
10-2
10-3
1.6 R2=0.998 1.4 1.2 1.0 0.2 0.4 0.6 0.8 1.0
0.4 0.3
-6
[DA]/μM
2x 10 5x 1 10 -5 0 -6
Absorbance (a.u.)
0.6
-8
10
-9
Bl an k
0.7
A0/A
C
10-7 5×10-7 10-6 5×10-6 10-5
10 -7 2x 10 -7 4x 10 -7 5x 10 -7 6x 10 -7 8x 1 10 -6 0 -7
Blank 10-9 10-8
0.2 0.1 400
500
600
700
800
900
Wavelength (nm)
Fig. 4. Colorimetric detection of concentrations of DA based on AuNPs (A) without and (B) with TGA2-, (C) the corresponding UV-vis absorption spectra of (B), and the inset of (C): absorbance ratio (A0/A) of (C) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 10-8, 10-7, 2×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 2×10-6, 5×10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
DA
+
+
Urea Oxa Fer Foli Gluc Lys Gly Lac UA Hist L-Tyr L-Asp Thr
Blank
K Ca2+ 2+ L-Try Zn + AA Na
NH4 CO 23 -
Cl
Fig. 5. Color photographs and Absorbance ratio (A580/A525) of the developed reagent with different analytes. Analytes: L-Tyrosine (L-Tyr), Lactate (Lac), Uric Acid (UA), L-Aspartic acid (L-Asp), Oxalate (Oxa), Histidine (Hist), Lysine (Lys), Threonine (Thr), Glucose (Gluc), Dopamine (DA), Glycine (Gly), Folic acid (Foli), Ferulic acid (Fer), Urea, Acrobatic Acid (AA), L-Tryptophan (L-Try), Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-. The concentrations of analytes are 10-6 M.
10-7
Blank
10-6
10-5
10-3
10-4
A
B 0.7
2.1 Blank -9
1.8
0.6
A0/A
Absorbance (a. u.)
10
-9
5x10 -8
10
-8
0.5
5x10
1.5 1.2
-7
10
-7
0.9
2x10
0.4
R2=0.990
-7
0.0 0.2 0.4 0.6 0.8 1.0
3x10
-7
4x10
0.3
[DA]/μM
-7
5x10
-7
6x10
-7
0.2
8x10 -6
10
-5
10
0.1 300
400
500
600
700
800
Wavelength (nm)
Fig. 6. (A) Colorimetric photographs and (B) UV-vis absorption spectra of the developed reagent with concentrations of DA in human urine dialysates. The inset: absorbance ratio (A0/A) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 5×10-9, 10-8, 5×10-8, 10-7, 2×10-7, 3×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
Blank
10-7
10-3
10-4
10-5
10-6
10-2
A
B
10
-9
5x10 10-8 5x10
0.6
10
-9
3x10 4x10 5x10 6x10
-8
1.2
-7
0.9
10
300
10
R2=0.988
0.0 0.3 0.6 0.9
-7
[DA]/μM
-7
-7 -7
8x10
0.2
1.5
-7
2x10
0.4
A0/A
0.8
Absorbance (a. u.)
1.8
Blank
-7
-6
-5
400
500
600
700
800
Wavelength (nm)
Fig. 7. (A) Colorimetric photographs and (B) UV-vis absorption spectra of the developed reagent with concentrations of DA in FBS dialysates. The inset: absorbance ratio (A0/A) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 5×10-9, 10-8, 5×10-8, 10-7, 2×10-7, 3×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
Table 1. Results for the determination of DA in human urine and FBS dialysates. spiked amount found amounta recoverya (µM) (µM) human urine 0 not detected 0.6 0.627 ± 0.006 104.5 ± 1.1 1.0 1.012 ± 0.010 101.3 ± 1.0 samples
FBS
a
Mean ± std, n = 3.
0 0.05 0.5
not detected 0.054 ± 0.002 0.521 ± 0.009
108.5 ± 4.0 104.2 ± 1.9
Graphical Abstract C
B
A
H O
H O
H O
H O S
HNH
HNH O
TGA2-
DA
H O
O HO
H O
O
H O O S
HNH HNH
DA
HO
H O
S TGA2-
O
O
HNH Colorimetric detection of dopamine (DA) based on AuNPs: (A) disperse AuNPs, (B) because of the covalent and noncovalent interactions between DA and AuNPs, AuNPs are aggregated and capped by DA (DA-AuNPs), (C) the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA induce AuNPs further aggregated.
Highlights >A low-cost, rapid, sensitive, and highly selective colorimetric method for dopamine (DA) detection was developed.>The limit of detection is lower than the lowest abnormal concentrations of DA in body fluid.>The developed method has been successfully applied for the detection of DA in urine and serum samples.
PII: DOI: Reference:
S0039-9140(15)00120-4 http://dx.doi.org/10.1016/j.talanta.2015.02.038 TAL15411
To appear in: Talanta Received date: 30 December 2014 Revised date: 17 February 2015 Accepted date: 20 February 2015 Cite this article as: Yumin Leng, Kun Xie, Liqun Ye, Genquan Li, Zhiwen Lu and Junbao He, Gold-nanoparticle-based colorimetric array for detection of dopamine in urine and serum, Talanta, http://dx.doi.org/10.1016/j.talanta.2015.02.038 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 galley proof before it is published in its final citable 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.
Gold-nanoparticle-based colorimetric array for detection of dopamine in urine and serum Yumin Leng1*, Kun Xie1, Liqun Ye2, Genquan Li1, Zhiwen Lu1, Junbao He1 1
College of Physics and Electronic Engineering, Nanyang Normal University, Nanyang 473061,
China. 2
College of Chemistry and Pharmaceutical Engineering, Nanyang Normal University, Nanyang
473061, China.
Corresponding Author *E-mail: [email protected] ABSTRACT: A highly selective method is presented for the colorimetric determination of dopamine (DA) using gold nanoparticles (AuNPs). DA caps on the surface of AuNPs (DAAuNPs) and induces the aggregation of AuNPs in alkaline solution. The DA-AuNPs are modified by the hydrolysate of thioglycolic acid (TGA2-) through Au-S bonds. The aggregation of AuNPs is accelerated by TGA2-, due to the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA. Upon the addition of DA, the solution shows a color change from red to purple (or yellow), which is also monitored to detect DA in human urine and fetal bovine serum samples. Here, the limits of colorimetric detection are as low as 10-7 M observed in Milli-Q water, urine and serum. Based on UV-vis absorption spectra, the limits of detection have been calculated to be 3.310-8 M, 1.010-7 M and 9.410-8 M in Milli-Q water, urine and serum, respectively. All the limits of detection are lower than the lowest abnormal concentrations of DA in urine (5.710-7 M) and blood (1.610-5 M). The good linear ranges from 0 to 10-6 M are used for the quantitative assay of DA in urine and serum samples. The applicability of our detection system is also verified by analysis of DA in urine and serum samples. The developed approach is without using complex financial instruments.
Keywords: Gold nanopartilces; colorimetric detection; dopamine; urine; serum 1. Introduction Dopamine (DA), a catecholamine derived from tyrosine, is an important neurotransmitter of the central and peripheral nervous systems [1,2]. It is widely distributed in the central neural system, brain tissues and body fluids of mammals and plays a vital role in the function of the central nervous, hormonal, renal and cardiovascular systems [3-6]. Normal level of DA in body supports blood pressure, fine motor activity, inspiration, intuition and focus, whereas abnormal DA concentrations in body often causes euphoria, addiction, obesity, even serious diseases such as schizophrenia, Parkinsonism, Alzheimer’s disease, Huntington’s disease, epilepsy, pheochromocytoma and neuroblastoma [3-10]. Moreover, hard drugs (sush as cocaine, opium, heroin, morphine, and codeine), doping, tobacco and alcohol can activate the secretion of DA in human body [11-15]. So it is necessary to determine the level of DA in human body fluid rapidly. To detect DA accurately, a number of physico-chemical methods such as fluorescence [16-18], electrochemical detection [19-21], and high performance liquid chromatography [22] have been reported. Although these methods are accurate, they are usually time-consuming, need complicated instruments or lack sensitivity. Thus, it still remains a great challenge to develop an inexpensive, fast and reliable method for determination of trace amount of DA in body fluid, such as blood and urine. Without the aid of expensive instruments, colorimetric method is convenient and can be easily monitored by the naked eyes [23-37]. Because of the unique distance-dependent optical property of AuNPs/silver nanoparticles (AgNPs), Au/AgNPs-based colorimeric assays have been widely used to detect analytes, such as ions, molecules and cell [27-37]. Au/AgNPsbased colorimeric methods for the detection of DA have been developed only recently [38-42]. For example, Qu group used the probe of AgNPs to detect DA in Milli-Q water [40]. To detect cerebral DA, Tian et al. designed AuNPs functionalized with two ligands [41]. Wang et
al. developed the functional AuNPs to determinate DA in human serum [42]. Although conceptually simple, the developed methods are usually limited by the low selectivity and the application in real systems. Therefore, substantial efforts are still needed for the development of efficient methods to meet the increasing demand for real DA detection with high selectivity. Herein, we develop a rapid, sensitive and selective approach for the colorimetric detection of DA using AuNPs, with theoretical simplicity and low technical demands. In alkaline solution, DA capps and aggregates AuNPs (DA-AuNPs) due to the covalent and noncovalent interactions between DA and AuNPs [43,44]. The DA-AuNPs are modified by the hydrolysate of thioglycolic acid (TGA2-) through Au-S bonds [30,34]. Due to the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA, the AuNPs are further aggregated [45,46]. With the increase of DA concentrations, the color of AuNPs gradually changes from an initial red to purple, then gray and finally yellow. The color changes are also monitored to detect DA in human urine and fetal bovine serum samples. The limits of detection (LOD) by the naked eyes are as low as 10-7 M in Milli-Q water and samples. The LODs by UV-vis spectroscopy are 3.310-8 M, 1.010-7 M and 9.410-8 M in Milli-Q water, urine and serum, respectively. All the LODs are lower than the lowest abnormal concentrations of DA and in urine (5.710-7 M) and blood (1.610-5 M) [47-49]. Moreover, the developed probe shows low response to 22 probable interferences comparing with DA. The interfering substances are considered comprehensively, comparising with those in the literature [38-42]. Therefore, the present method shows high selectivity for DA detection, and has been successfully applied for the detection of DA in urine and serum samples. 2. Experimental section 2.1 Materials and Instrumentation Chloroauric acid tetrahydrate (HAuCl4•4H2O), hexadecyl trimethyl ammonium bromide (CTAB), sodium borohydride (NaBH4), NaCl, KCl, Na2CO3, CaCl2, ZnCl2, NH4Cl, lactate,
uric acid, urea, oxalate, histidine, lysine, urea, and ascorbic acid were obtained from Sinopharm Chemical Reagent Co., Ltd (Beijing, China). Thioglycolic acid (TGA), FeCl2•4H2O, L-tyrosine, L-aspartic acid, threonine, glucose, dopamine hydrochloride, glycine, folic acid, ferulic acid and tryptophan were obtained from Aladdin-regent Co. Ltd (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, USA). All chemicals were used as received without further purification. Millipore-Q water was used throughout the experiment. All glassware were washed with aqua regia (HCl/HNO3 = 3:1 (v/v)) and then cleaned with Milli-Q water. Transmission electron microscopy (TEM) was performed on a Tencai F20 instrument with an acceleration voltage of 200 kV. UV-vis absorption spectra were recorded using a Lambda 950 UV-vis spectrophotometer from Perkin Elmer. 2.2 Preparation of AuNPs AuNPs were prepared through the reduction of HAuCl4 by NaBH4 in the presence of CTAB, according to Murphy's method [50]. In brief, 5 mL of 5 mM HAuCl4 solution was added into 95 mL double distilled water with vigorous stirring. 1 mL of 20 mM CTAB was injected into the mixture under vigorous stirring. Then, 1.5 mL of 0.1 M freshly prepared NaBH4 solution was added slowly during stirring. The reaction was kept for 30 min, during the time the color changed from pink to deep red. The prepared AuNPs solution was stored at 4 0C until use. 2.3 Colorimetric Detection of DA 2.3.1 Sensitivity To evaluate the detectable minimum concentration of DA, the typical colorimetric analysis was carried out by following steps. Firstly, 30 μL of DA solutions with different concentrations were mixed with 1380 μL AuNPs. Next, 30 μL NaOH (5 M) was added to the above mixture, 10 min later, 60 μL of 1% TGA was added, and the mixture was allowed to
react for 20 min at 25 0C. Color change was observed with the naked eye and UV-vis absorption spectra were recorded with a UV-vis spectrophotometer. 2.3.2 Selectivity The selectivity for DA was investigated as follows: 30 μL of 5×10-5 M the representative biomolecules and ions that probably exist in urine and serum, such as L-tyrosine (L-Tyr), lactate (Lac), uric acid (UA), L-aspartic acid (L-Asp), oxalate (Oxa), histidine (Hist), lysine (Lys), threonine (Thr), glucose (Gluc), glycine (Gly), folic acid (Foli), ferulic acid (Fer), urea, acrobatic acid (AA), L-tryptophan (L-Try), DA, Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-, were added into 1380 μL AuNPs, respectively. Next, 30 μL NaOH (5 M) was added into the above mixture, 10 min later, 60 μL of 1% TGA was added, and the mixture was allowed to react for 20 min at 25 0C. The final concentrations of analytes are 10-6 M. The corresponding photo images and UV-vis absorption spectra were recorded. 2.3.3 Practical application In order to assess the validity of the developed procedure, the method was applied to detect DA in biological fluids such as human urine and FBS samples. The spiked samples were obtained by adding DA with different concentration to the dialysates of human urine and FBS. The detection procedure of DA in biological fluids is similar with that in double distilled water. The corresponding photo images and UV-vis absorption spectra were recorded. 3. Results and discussion 3.1. Principle of DA Detection Scheme 1 shows the probable mechanism for the detection of DA. Due to the strong covalent and noncovalent interactions between DA and AuNPs [43,44], DA caps and aggregates AuNPs (DA-AuNPs) in alkaline solution (See Scheme 1B). The DA-AuNPs are modified by TGA2- ([SCH2CO2]2-) through Au-S bonds [30,34]. Due to the strong hydrogenbonds (NH···OC and OH···OC) formed between TGA2- and DA [45,46], the AuNPs are further aggregated (See Scheme 1C). Although the exact function of alkaline is unknown at
this time, it is likely to provide strong oxidizing environment, hence speeds up the reaction in the system. Moreover, under oxidizing conditions, DA reacts with thiols via Schiff base reaction [51]. Scheme 1 TEM characterizations are used to elucidate the principle of DA detection. TEM images (See Fig. S1) show the microstructures of the AuNPs in the absence and presence of 10-6 M DA and TGA2-. Notably, without DA, AuNPs are well monodispersed (See Fig. S1, panel A). Nevertheless, in the presence of 10-6 M DA, AuNPs start to aggregate due to the covalent and noncovalent interactions between DA and AuNPs [43], and DA capps on the surface of AuNPs (DA-AuNPs) in alkaline solution [44]. The aggregation of DA-AuNPs is accelerated by the inducer of TGA2- (See Fig. S1, panel C), because of the strong hydrogen-bond interactions between TGA2- and DA [45,46]. Meanwhile, DLS experimental results show that the sizes of AuNPs are also increased with the increase of DA concentration (See Fig. 1). Fig. 1 3.2 Optimization of the Expremental Parameters The performance of the as-developed method for DA detection may be strongly influenced by the experimental conditions such as pH of the colloidal solution, reaction temperature, and reaction time. Thus, each detection parameter was optimized in our study, while keeping other parameters constant. The effect of pH on the colorimetric detection of DA was investigated by adding different concentrations of NaOH, while keeping DA concentration unchanged (10-6 M). Comparing with the solution without DA (blank), the color change of the solution with 10-6 M DA can be immediately observed when the concentration of NaOH is beyond 5 M (See Fig. S2). At this time, the pH of the solution was measured to be 13, which was chosen in the following experiments.
As expected, the reaction temperature has great effect on DA detection. The UV-vis absorption spectra of the developed reagent in the absence and presence of 10-5 M DA, and the corresponding absorption ratio (Ablank/A[DA]=10-5 M) at different temperature are shown in Fig. 2. As shown in Fig. 2A, the UV-vis absorption peak of the developed reagent is redshifted when the reaction temperature is above 25 0C, indicating that AuNPs are unstable and aggregated at higher temperature (> 25 0C). Moreover, with the increase of temperature, the absorption ratio (Ablank/A[DA]=10-5 M) increases quickly before 25 0C, and slowly when the reaction temperature is above 25 0C (See Fig. 2, panel C). Thus, 25 0C was chosen as the optimal reaction temperature in the experiments. Fig. 2 The incubation time also influences the colorimetric response of the developed reagent in the presence of DA. To investigate the effect of reaction time, the changes of the UV-vis absorption spectra of the developed reagent with 10-6 M DA were recorded within 35 min and the absorption ratio of the minimum and maximum (Amin/Amax) were obtained, as shown in Fig. 3. The value of Amin/Amax increases exponentially with the increase of reaction time (See Fig. 3, panel B), and reaches an equilibrium almost after 20 min. Therefore, 20 min was chosen for the optimal incubation time in the experiments. Fig. 3 3.3 Colorimetric Detection of DA Using the Developed Method 3.3.1 Sensitivity In the absence of TGA2-, the limit of colorimetric detection is approximately 510-6 M (See Fig. 4, panel A), which is higher than the lowest abnormal concentration (5.710-7 M) of DA in urine [47]. In the presence of TGA2- and upon addition of increasing concentrations of DA, the color of AuNPs gradually changes from initially red to purple, then gray and finally yellow. And the limit of colorimetric detection is significantly proved to be 10-7 M (See Fig. 4, panel B), which is lower than the previously reported data (1.85×10-7 M) [41] (See Table S1
in the Supporting Information). This observation reveals that the sensitivity for DA detection can be greatly imporved by adding TGA2-. These results are further confirmed by UV-vis spectroscopy. The UV-vis absorption spectra of the developed reagent with different concentrations of DA are shown in Fig. 4, panel C. The absorption peak is clearly red-shifted and the absorption intensity decreases with the increase of DA concentration. The ratio (A0/A) of the absorption intensity (A0 and A are corresponding to the developed reagent with concentrations of DA (0 and 10-9 ~ 10-5 M).), was used to express the molar ratio between dispersed and aggregated AuNPs. The ratio (A0/A) as a function of DA concentration is shown in Fig. 4C inset. The linear relationship (R2=0.998) can be inferred, manifesting the dynamic range from 0 to 10-6 M and the LOD of 3.3×10-8 M (See the calculation method in the Supporting Information). As shown in Table S1 (see the Supporting Information), the linear range of this approach is wider than those of other AuNPs as colorimetric probes [41, 42]. The LOD of 3.3×10-8 M is lower than the reported data (5×10-8 M and 7×10-8 M) in the literature [41,42]. Moreover, the detection limits of the colorimetric assay and calculation for DA (10-7 M and 3.3×10-8 M) in Milli-Q water are lower than the lowest abnormal concentrations of DA (5.710-7 M and 1.610-5 M) in urine and blood [47-49]. Thus, the present strategy developed in this study is sensitive and able to realize the colorimetric detection of DA in real samples, such as urine and blood. Fig. 4 3.3.2 Selectivity The complexity of urine and serum presents a great challenge to the analytical methods for DA detection not only in the detection limit and sensitivity but, more importantly, in selectivity. The selective recognition of DA by the developed method was investigated by considering other 22 biomolecules and ions that probably exist in urine and serum, such as LTyr, Lac, UA, L-Asp, Oxa, Hist, Lys, Thr, Gluc, Gly, Foli, Fer, urea, AA, L-Try, Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-. The interfering substances are considered comprehensively,
comparising with those in the literature [38-42]. The concentrations of analytes are 10-6 M. The corresponding photo image and UV-vis absorption ratio (A580/A525) are shown in Fig. 5. It is clear that the developed probe has very high specificity toward DA (See Fig. 5). Fig. 5 3.3.3 Practical application As demonstrated above, the present colorimetric method for DA detection with high selectivity provides a direct platform for assaying DA in urine and serum. Thus, the developed method was applied in detection of DA in biological fluids such as human urine and FBS samples. The spiked samples were obtained by adding concentrations of DA to the dialysates of human urine and FBS. The corresponding photo images and UV-vis absorption spectra are shown in Fig. 6 and Fig. 7, respectively. The color of the solutions changes from red to purple, gray and yellow with the increase of DA concentrations (See Fig. 6 and Fig. 7, panels A). Although the complex elements in the spiked samples slightly affect the color changes (compared with Fig. 4, panel B), the limits of colorimetric detection are both up to 10-7 M. UV-vis spectroscopy provides a more precise approach for the detection of DA contained in the dialysates of human urine and FBS (See Fig. 6 and Fig. 7, panels B). As the contained concentrations of DA increase, the absorption peak is red-shifted and the absorption intensity decreases. The ratio (A0/A) as a function of DA concentration are shown in Fig. 6 and Fig. 7 B insets, respectively. The linear relationships can be inferred, manifesting the dynamic ranges from 0 to 10-6 M and the LODs of 1.0×10-7 M and 9.4×10-8 M (See the calculation method in the Supporting Information), respectively. The detection limits of the colorimetric assays (both 10-7 M) and calculations (1.0×10-7 M and 9.4×10-8 M) for DA in the dialysates of human urine and FBS are lower than the lowest abnormal DA concentrations of 5.710-7 M (urine) and 1.610-5 M (blood) [47-49], respectively. As shown in Table S1 (see the Supporting Information), we first present the analytical parameters of linearity and sensitivity in real application, comparing with those of other AuNPs as colorimetric probes
[41,42]. Moreover, the accuracy and precision of the developed procedure were assessed, as shown in Table 1. The found DA concentrations using the developed method are in good agreement with the spiked amount. The recoveries are within the range from 101.3 to 108.5%. Thus, the developed method has been successfully applied for the detection of DA in urine and serum samples. 4. Conclusions In summary, we have developed a rapid, sensitive, and highly selective strategy for the colorimetric visualization of DA. Because of the strong covalent and noncovalent interactions between DA and AuNPs, and the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA, so we observed that DA capped and aggregated AuNPs, and the aggregation was accelerated by TGA2-. The selectivity of the DA detection system by the naked eyes and UV-vis absorption spectra is excellent comparing with 22 probable interferences. The interfering substances are considered comprehensively, and we first present the analytical parameters of linearity and sensivity in real application, comparising with those in the literature. The limits of colorimetric detection have been determined to be 10-7 M in Milli-Q water, urine and serum. The LODs have been calculated to be 3.310-8 M (Milli-Q water), 1.0×10-7 M (urine) and 9.4×10-8 M (serum), respectively. All the LODs are lower than the lowest abnormal DA concentrations in urine (5.710-7 M) and blood (1.610-5 M). The good linear relationship between A0/A and DA concentration from 0 to 10-6 M, which are used for the quantitative assay of DA in urine and serum samples. These results indicate that the developed detection system is applicable for rapid colorimetric detection of DA with excellent selectivity and high sensitivity in urine and serum samples. Acknowledgements This work was supported by the Science and Technology Program of Education Department of Henan Province (No. 14A430024), Natural Science Foundation of Nanyang Normal University (Nos. zx2014087), National Natural Science Fund (E041604/51374132).
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C
B
A
H O
H O
H O
H O S
HNH
HNH O
TGA2-
DA
H O
O
H O
H O
O
H O O S
HNH HNH
DA
HO
H O
S
TGA2-
O
O
HNH
Scheme 1. Colorimetric detection of dopamine (DA) based on AuNPs: (A) disperse AuNPs, (B) AuNPs aggregated and capped by DA (DA-AuNPs), (C) AuNPs further aggregated by TGA2- ([SCH2CO2]2-).
A
20
[DA]/0M
Number (%)
Number (%)
25 20 15 10 5 0
[DA]/10 M
15 10 5 0
0
500
1000 1500 2000
0
500
Size (d.nm) [DA]/10 M
20 15 10 5 0 0
25
-6
C
500
1000 1500 2000
Size (d.nm)
1000 1500 2000
Number (%)
25
Number (%)
-7
B
-2
[DA]/10 M
D
20 15 10 5 0 0
500
Size (d.nm)
1000 1500 2000
Size (d.nm)
Fig. 1. Size distribution of the AuNPs measured by DLS in the (A) absence and presence of (B) 10-7 M, (C) 10-6 M, and (D) 10-2 M DA induced by TGA2-.
Absorbance (a. u.)
0.8
0
Temp. ( C)
A
0 10 15 20 25 30 35 40
0.6 0.4 0.2 400
500
600
700
800
Wavelength (nm)
Absorbance (a. u.)
0.7
0
Temp. ( C)
B
0 10 15 20 25 30 35 40
0.6 0.5 0.4 0.3 0.2 400
500
600
700
800
Wavelength (nm) 40
C
Temp. (0C)
30 20 10 0 1.0
1.2
1.4
1.6
1.8
2.0
2.2
Ablank/A[DA]=10-5M
Fig. 2. UV-vis absorption spectra of the developed reagent at different temperature in the (A) absence and (B) presence of 10-5 M DA. (C) Temperature-absorbance ratio of Ablank/A[DA]=10-5 M.
Absorbance (a. u.)
Time (min)
A
0.7
0 1 3 6 9 12 15 18 21 24 27 30 35
0.6 0.5 0.4 0.3 0.2 0.1 400
500
600
700
800
900
Wavelength (nm) 0.70
B
Amin/Amax
0.69 0.68 0.67 0.66 0.65 -5
0
5
10
15
20
25
30
35
40
Time (min) Fig. 3. (A) Time-dependent absorption spectra for the developed reagent containing 10-6 M DA. (B) Time-absorbance ratio of Amin/Amax.
Blank 10-9
10-8
10-7
10-6
10-4
5×10-6 10-5
10-3
10-2
A
B
10
0.5
10-4
10-2
10-3
1.6 R2=0.998 1.4 1.2 1.0 0.2 0.4 0.6 0.8 1.0
0.4 0.3
-6
[DA]/μM
2x 10 5x 1 10 -5 0 -6
Absorbance (a.u.)
0.6
-8
10
-9
Bl an k
0.7
A0/A
C
10-7 5×10-7 10-6 5×10-6 10-5
10 -7 2x 10 -7 4x 10 -7 5x 10 -7 6x 10 -7 8x 1 10 -6 0 -7
Blank 10-9 10-8
0.2 0.1 400
500
600
700
800
900
Wavelength (nm)
Fig. 4. Colorimetric detection of concentrations of DA based on AuNPs (A) without and (B) with TGA2-, (C) the corresponding UV-vis absorption spectra of (B), and the inset of (C): absorbance ratio (A0/A) of (C) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 10-8, 10-7, 2×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 2×10-6, 5×10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
DA
+
+
Urea Oxa Fer Foli Gluc Lys Gly Lac UA Hist L-Tyr L-Asp Thr
Blank
K Ca2+ 2+ L-Try Zn + AA Na
NH4 CO 23 -
Cl
Fig. 5. Color photographs and Absorbance ratio (A580/A525) of the developed reagent with different analytes. Analytes: L-Tyrosine (L-Tyr), Lactate (Lac), Uric Acid (UA), L-Aspartic acid (L-Asp), Oxalate (Oxa), Histidine (Hist), Lysine (Lys), Threonine (Thr), Glucose (Gluc), Dopamine (DA), Glycine (Gly), Folic acid (Foli), Ferulic acid (Fer), Urea, Acrobatic Acid (AA), L-Tryptophan (L-Try), Zn2+, K+, Ca2+, Na+, NH4+, Cl-, and CO32-. The concentrations of analytes are 10-6 M.
10-7
Blank
10-6
10-5
10-3
10-4
A
B 0.7
2.1 Blank -9
1.8
0.6
A0/A
Absorbance (a. u.)
10
-9
5x10 -8
10
-8
0.5
5x10
1.5 1.2
-7
10
-7
0.9
2x10
0.4
R2=0.990
-7
0.0 0.2 0.4 0.6 0.8 1.0
3x10
-7
4x10
0.3
[DA]/μM
-7
5x10
-7
6x10
-7
0.2
8x10 -6
10
-5
10
0.1 300
400
500
600
700
800
Wavelength (nm)
Fig. 6. (A) Colorimetric photographs and (B) UV-vis absorption spectra of the developed reagent with concentrations of DA in human urine dialysates. The inset: absorbance ratio (A0/A) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 5×10-9, 10-8, 5×10-8, 10-7, 2×10-7, 3×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
Blank
10-7
10-3
10-4
10-5
10-6
10-2
A
B
10
-9
5x10 10-8 5x10
0.6
10
-9
3x10 4x10 5x10 6x10
-8
1.2
-7
0.9
10
300
10
R2=0.988
0.0 0.3 0.6 0.9
-7
[DA]/μM
-7
-7 -7
8x10
0.2
1.5
-7
2x10
0.4
A0/A
0.8
Absorbance (a. u.)
1.8
Blank
-7
-6
-5
400
500
600
700
800
Wavelength (nm)
Fig. 7. (A) Colorimetric photographs and (B) UV-vis absorption spectra of the developed reagent with concentrations of DA in FBS dialysates. The inset: absorbance ratio (A0/A) vs. DA concentrations. (The concentrations of DA: 0, 10-9, 5×10-9, 10-8, 5×10-8, 10-7, 2×10-7, 3×10-7, 4×10-7, 5×10-7, 6×10-7, 8×10-7, 10-6, 10-5, 10-4, 10-3 and 10-2 M, respectively.)
Table 1. Results for the determination of DA in human urine and FBS dialysates. spiked amount found amounta recoverya (µM) (µM) human urine 0 not detected 0.6 0.627 ± 0.006 104.5 ± 1.1 1.0 1.012 ± 0.010 101.3 ± 1.0 samples
FBS
a
Mean ± std, n = 3.
0 0.05 0.5
not detected 0.054 ± 0.002 0.521 ± 0.009
108.5 ± 4.0 104.2 ± 1.9
Graphical Abstract C
B
A
H O
H O
H O
H O S
HNH
HNH O
TGA2-
DA
H O
O HO
H O
O
H O O S
HNH HNH
DA
HO
H O
S TGA2-
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HNH Colorimetric detection of dopamine (DA) based on AuNPs: (A) disperse AuNPs, (B) because of the covalent and noncovalent interactions between DA and AuNPs, AuNPs are aggregated and capped by DA (DA-AuNPs), (C) the strong hydrogen-bonds (NH···OC and OH···OC) formed between TGA2- and DA induce AuNPs further aggregated.
Highlights >A low-cost, rapid, sensitive, and highly selective colorimetric method for dopamine (DA) detection was developed.>The limit of detection is lower than the lowest abnormal concentrations of DA in body fluid.>The developed method has been successfully applied for the detection of DA in urine and serum samples.
