Accepted Manuscript Gold nanoparticles-based chemiluminescence resonance energy transfer for ultrasensitive detection of melamine Jianxiu Du, Yadi Wang, Weimin Zhang PII: DOI: Reference:

S1386-1425(15)00539-9 http://dx.doi.org/10.1016/j.saa.2015.04.067 SAA 13618

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

27 October 2014 10 March 2015 22 April 2015

Please cite this article as: J. Du, Y. Wang, W. Zhang, Gold nanoparticles-based chemiluminescence resonance energy transfer for ultrasensitive detection of melamine, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.04.067

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Gold nanoparticles-based chemiluminescence resonance energy transfer for ultrasensitive detection of melamine

Jianxiu Du *, Yadi Wang, Weimin Zhang Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China *Corresponding author. Tel.: +86 29 81530726; Fax: +86 29 81530727. Email address: [email protected]

ABSTRACT A turn-on chemiluminescence resonance energy transfer method was fabricated for the determination of melamine by using bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide-fluorescein chemiluminescence reaction as a donor and dispersed gold nanoparticles

as

an

acceptor.

The

chemiluminescence

signal

of

bis(2,4,6-trichlorophenyl)oxalate-hydrogen peroxide-fluorescein reaction decreased significantly in the presence of dispersed gold nanoparticles because the absorption band of dispersed gold nanoparticles perfectly overlapped with the chemiluminescence spectrum. Melamine could induce the aggregation of gold nanoparticles, leading to a dramatic red-shift of the absorption band of dispersed gold nanoparticles. The absorption band of the aggregated gold nanoparticles doesn’t overlap with the

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chemiluminescence spectrum of the reaction. In such a case, chemiluminescence resonance energy transfer couldn’t happen and the chemiluminescence signal was restored. The procedure allowed the measurement of 3.2×10 -12-3.2×10 -7 mol/L melamine with a limit of detection of 3×10-13 mol/L. The method was applied to the determination of melamine in spiked milk samples; with recoveries within the range 94.1-104.2%. Keywords: Melamine; Chemiluminescence; Resonance energy transfer; Gold nanoparticles

1. Introduction Melamine, an organic nitrogenous compound with a 1,3,5-triazine skeleton, finds wide use in the production of plastics, dyes, fertilizers, and fabrics. Due to its high nitrogen contents (66% by mass), melamine may have been unlawfully adulterated to milk to boost the apparent assay results of protein content by Kjeldahal method [1] and Dumas method [2]. Melamine contamination has been reported in products such as milk, infant formula, frozen yogurt, biscuits, candy, coffee drinks, and pet food [3]. The toxicity of melamine has caught much attention since the outbreak of nephrolithiasis among children ingesting melamine-contaminated infant formula in China [4, 5]. Melamine has become an issue of widespread food safety concern [6, 7]. The United Nation’s food standards body, Codex Alimentarius Commission, has issued a maximum allowable amount of melamine at 1 mg/kg in powdered infant formula and 2.5 mg/kg for other

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foods and animal feed [8]. The Chinese Department of Health has enacted the upper limits of melamine content in dried infant formula, liquid milk and dairy products (with >15% milk content) at 1 mg/kg, 2.5 mg/kg and 2.5 mg/kg, respectively [9]. Therefore, there is an urgent need for the analysis of melamine in the milk products, eggs and animal feeds to detect economically motivated adulteration or unintentional contamination. The standard methods enacted by Chinese government for determining melamine in raw milk and dairy products include high performance liquid chromatography with ultraviolet detection (HPLC-UV), liquid chromatography with mass spectrometry detection (LC-MS), and gas chromatography with mass spectrometry detection (GC-MS) [10]. The limits of quantification are of 2 mg/kg, 0.01 mg/kg, and 0.05 mg/kg for HPLC, LC-MS, and GC-MS, respectively. However, the high cost of operation and maintenance of GC/LC-MS systems as well as the derivatization of GC-MS limit their use in production facilities and routine analysis. Other reported analytical methods included spectrophotometry [11, 12], fluorescence [13, 14], and electrochemical method [15, 16]. Gold nanoparticles (AuNPs)-based colorimetry for detection of melamine was also proposed, based upon the fact that it could induce the aggregation of AuNPs, causing dramatic and visible color changes [17-20]. AuNPs-based colorimetric assays generally had narrow linear range and relatively low sensitivity; the detection limits were usually at ppb level. Chemiluminescence (CL) detection with the advantages of high sensitivity and wide linear dynamic range has also used for the analysis of

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melamine [21-25]. Table 1 summarizes the analytical performances of the previous reported CL methods for the determination of melamine. We here proposed a new strategy for the CL determination of melamine. The method was based on the inner effect of AuNPs on bis(2,4,6-trichlorophenyl)oxalate (TCPO)-hydrogen peroxide-fluorescein system, which led to a significant decrease in CL signal. Melamine could induce the aggregation of AuNPs [17-20], causing a red-shift of absorption band of AuNPs. Thus, the energy transfer between TCPO-hydrogen peroxide-fluorescein CL reaction and AuNPs could not happen in the presence of melamine. As a result, the CL intensity was restored. The procedure was simple, sensitive, selective, and applied to the determination of melamine in spiked liquid milk products. 2. Experimental 2.1. Apparatus CL measurements were carried out on an IFFM-E CL analyzer (Xi’an Remex, China). Absorption spectra were taken on a TU-1901 spectrophotometer (Purkije General, China).

Fluorescence

spectra

were

recorded

on

an

F-7000

fluorescence

spectrophotometer (Hitachi, Japan). Transmission electron microscopy (TEM) images were measured by using a JEM–2100 transmission electron microscope(Japan Electronic Company, Japan). 2.2. Chemicals All chemicals were of analytical grade and distilled de-ionized water was used

4

throughout the experiments. Melamine was purchased from Tianjin Fuchen Chemical Reagents Factory (Tianjin, China). Bis(2,4,6-trichlorophenyl)oxalate (TCPO) was purchased

from

Beijing

J&K

Scientific

Ltd

(Beijing,

China).

Hydrogen

tetrachloroaurate tetrahydrate (HAuCl4•4H2O) was purchased from Shanghai No. 1 Chemical Reagent Co., Ltd (Shanghai, China). Other chemicals were obtained from Xi’an Chemical Reagent Co. (Xi’an, China). Melamine stock solution (5.0×10

-3

mol/L) was prepared by dissolving 63.1 mg of

melamine in 100 mL of water. Working solutions of melamine were prepared by dilution of the stock solution with water before use. All melamine solutions were protected from light and stored in a refrigerator. TCPO solution (3.0×10 -4 mol/L) was prepared with acetone. Fluorescein solution (2.0×10 -5 mol/L) was prepared with 0.05 mol/L tris–HCl buffer (pH 7.5). Hydrogen peroxide solution (0.6 mol/L) was freshly prepared by the dilution of 30% hydrogen peroxide with water. 2.3. Preparation of citrate–stabilized AuNPs Citrated–stabilized AuNPs were synthesized by classic Frens method [26] with minor modification. Briefly, sodium citrate solution (2%, 10.0 mL) was rapidly added in boiling HAuCl4 solution (0.08%, 100.0 mL) under vigorous stirring. The solution changed from pale yellow to purple, finally to wine-red. After continually boiling for additional 20 min, the heat source was removed and the solution was continually stirred until to room temperature. The maximum absorption wavelength of the prepared AuNPs solution located at 522 nm and the sizes was 13.0±3.6 nm. The concentration of the

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prepared AuNPs was calculated to be 24 nmol/L with the formula suggested by Cumberland and Strouse [27]. 2.4. Procedure In the CL reaction cell, 80.0 µL of melamine solution, 20.0 µL of the as-prepared AuNPs solution, and 50.0 µL of fluorescein solution (2.0×10-5 mol/L) were successively added and mixed. After incubating at room temperature for 10 min, 50.0 µL of hydrogen peroxide solution (0.6 mol/L) was added into above mixture. The CL reaction was activated by injecting 50.0 µL of TCPO solution (3.0×10 -4 mol/L) into the reaction cell. The CL signal produced was monitored via a CR 105 photomultiplier tube (Hamamatsu Photonics (China) Co., Ltd), biased at an operating voltage of 600 V. The determination of melamine was based on the restored CL signal (∆I, peak height), calculated as ∆I=Is-Ib, where Is was the CL signal in the presence of melamine and Ib was the blank. 3. Results and discussion 3.1. Principle for melamine detection using AuNPs-based CRET The reaction of TCPO with hydrogen peroxide generates unstable 1,2-dioxetanedione, which transfers the energy to fluorescein molecules to form electronically excited fluorescein. The excited fluorescein decays to the ground state, producing the CL emission at 514 nm (Fig. 1a). Dispersed AuNPs solution is wine-red in color and has a maximum absorption wavelength at 522 nm (Fig. 1b), which matches well with the emission spectrum of TCPO-hydrogen peroxide-fluorescein CL system. When

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dispersed AuNPs was added into TCPO-hydrogen peroxide-fluorescein CL system, the excited fluorescein molecules transfer their energy to non-radiation dispersed AuNPs, causing a decrease in CL signal (Fig. 2b). As reported previously, melamine had a strong electrostatic interaction with citrate-capped AuNPs, which decreased the stability of the AuNPs, inducing the aggregation of AuNPs [17, 18]. The TEM images of AuNPs solution alone and the mixture of AuNPs with melamine was shown in Fig. 3. The color of the solution changed from wine-red to purple, concomitant with a significant decrease in the absorbance at 522 nm and a red-shift of the maximum absorption band of the AuNPs to 728 nm (Fig. 1c). Since the absorption spectrum of the aggregated AuNPs no longer overlapped with the emission spectrum of TCPO-hydrogen peroxide-fluorescein CL system, the CRET couldn’t occur. As a result, the CL signal was restored (Fig. 2c). 3.2. Optimization of detection conditions The CL conditions of TCPO-hydrogen peroxide-fluorescein system were investigated elsewhere [28], namely 0.12 mol/L hydrogen peroxide, 60.0 µmol/L TCPO, and 4.0 µmol/L fluorescein (in 0.05 mol/L tris- HCl buffer, pH 7.5), we here thoroughly examined the effect of the concentration of AuNPs and the incubation time of AuNPs with melamine on the determination of melamine. The concentration of AuNPs is critical on the determination of melamine attributing to its influence on the energy transfer efficiency between CL reaction and AuNPs and the aggregation of AuNPs induced by melamine. As shown in Fig. 4, in the absence of

7

AuNPs, melamine has a very slightly inhibitory effect on the TCPO-hydrogen peroxide-fluorescein system. As increasing the concentration of AuNPs from 0.48 nmol/L to 1.92 nmol/L, the restored CL intensity increased with increasing the concentration of AuNPs. Further increase in the concentration of AuNPs from 1.92 nmol/L to 4.8 nmol/L caused a decrease in the restored CL intensity. Thus, 1.92 nmol/L was selected as the optimum concentration of AuNPs. The incubation time of AuNPs and melamine is another important factor that affected the detection of melamine. Fig. 5 shows the restored CL intensity as a function of incubation time. The restored CL intensity increased with the increase of incubation time up to 7 min, over which, the restored CL intensity started to remain constant. This result was consistent with that of Li’s work, colorimetric determination of melamine with AuNPs [18]. Finally, 10 min of the incubation time was employed. 3.3. Analytical figures Calibration graph for melamine was prepared under the conditions described above. A linear calibration plot was obtained for melamine over the range 3.2×10-12-3.2×10-7 mol/L. The linear regression equation was ∆I=94.03 lg c(10-12mol/L)+42.55 with a correlation coefficient of 0.998 (n=9). The relative standard deviation was 3.3% for eleven replicate determination of 3.2×10 -10 mol/L melamine solution. The limit of detection, based on three times the standard deviations of blank (n=11) was determined to be 3×10-13 mol/L. As can be seen from Table 1, this method exhibits excellent performance in terms of

8

wider linear range and lower detection limit in contrast with the previous reported CL methods for the determination of melamine [21-25]. 3.4. Selectivity To evaluate the selectivity of the method toward to the determination of melamine, the responses of this method to melamine (3.2×10-9 mol/L) and to other potentially interfering substances were investigated. As shown in Fig. 6, other substances, including 3.2×10-8 mol/L Ca2+, Mg2+, Zn2+, SO42-, glucose, lactose, 3.2×10-7 mol/L K+, Na+, Fe3+, Cl-, NO3-, and 1.6×10 -6 mol/L sucrose couldn’t produce a measurable CL signal, indicating that the assay had an excellent selectivity for melamine. 3.5. Determination of melamine in milks To evaluate the practical application of the method, it was applied to the determination of melamine in milk samples. Milk samples from four different manufacturers were purchased from a local market. A known amount of melamine was added into 0.50 mL of milk samples. Proteins in the samples were removed by adding 2.0 mL of acetonitrile into the milk samples [29]. After centrifuging at 10000 rpm for 30 min. the supernatant was filtered through a 0.22 µm membrane to remove lipids. Then, acetonitrile was evaporated by the flow of nitrogen; the residual was dissolved into 50 mL of water for analysis. The blank milk samples without adding melamine were treated with the same procedure. The results are listed in Table 2. The percentage recovery of melamine in milk samples ranged from 94.1% to 104.2%, which showed the method had reliable recovery.

9

4. Conclusion A turn-on CRET sensing method was developed for the determination of melamine based on CRET between TCPO-hydrogen peroxide-fluorescein system and AuNPs. The presence of melamine induced the aggregation of AuNPs, reducing the CRET process and restoring the CL signal. The procedure has several advantages, including high sensitivity (The detection limit was 3×10-13 mol/L), wider linear dynamic range (six orders of magnitude, 3.2×10-12-3.2×10 -7 mol/L), and high specificity. Moreover, this CRET detection platform can be easily explored to determine other analytes that can induce the aggregation of AuNPs. Acknowledgements The authors acknowledge supports from the Natural Science Foundation of Shaanxi Province (2014JM2037) and Fundamental Research Funds for the Central Universities (GK201302014, GK261001305). References [1] ISO 8968-1-2014, Milk and milk products-Determination of nitrogen content-Part I: Kjeldahl principle and crude protein calculation. [2] ISO 14891-2002, Milk and milk products-Determination of nitrogen content-Routine method using combustion according to the Dumas principle. [3] World Health organization, Melamine contamination event, China, 2008, retrieved December 2, 2008, from http://www.who.int/foodsafety/fa management/infosan events/en/index.html.

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[4] N. Guan, Q. Fan, J. Ding, J. Lu, Y. Ai, G. Xu, S. Zhu, C. Yao, L. Jiang, J. Miao, H. Zhang, D. Zhao, X. Liu, Y. Yao, New Engl. J. Med. 360 (2009) 1067-1074. [5] K. Hau, T.H. Kwan, P.K. Li, J. Am. Soc. Nephrol. 20 (2009) 245-250. [6] C.Y. Zenobia, W.F. Chan, Trends Food Sci. Tech. 20 (2009) 366-373. [7] F.X. Sun, W. Ma, L.G. Xu, Y.Y. Zhu, L.Q. Liu, C.F. Peng, L.B. Wang, H. Kuang, C.L. Xu, TRAC-Trend Anal. Chem. 29 (2010) 1239-1249. [8] Food and Agriculture Organization of the United Nations, International experts limit

melamine

levels

in

food,

retrieved

July

6,

2010,

from

http://www.fao.org/news/story/en/item/43719/icode/ [9] General Administration of Quality Supervision, Inspection and Quarantine of the People’s

Republic

of

China,

from

retrieved

October

8,

2008,

http://www.aqsiq.gov.cn/zjxw/zjxw/zjftpxw/200810/t20081008_926.htm. [10] Chinese Standards, Determination of melamine in raw and dairy products, GB/T 22388-2008. [11] C. Li, Y.F. Li, B. Liang, N. Li, Z.Z. Li, Anal. Methods 5 (2013) 5760-5766. [12] W. Chansuvan, S. Panich, A. Imyim, Spectrochim. Acta, A 13 (2013) 154-158. [13] J. Rima, K. Assaker, F. El Omar, C.B. Karim, Talanta 116 (2013) 277-282. [14] X.Y. Cao, F. Shen, M.W. Zhang, J.J. Guo, Y.L. Luo, J.Y. Xu, Y. Li, C.Y. Sun, Dyes Pigments 111 (2014) 99-107. [15] W.R. de Araujo, T. Paixao, Electrochim. Acta 117 (2014) 379-384. [16] G.L. Xu, H.L. Zhang, M. Zhong, T.T. Zhang, X.J. Lu, X.W. Kan, J. Electroanal.

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Chem. 713 (2014) 112-118. [17] K.L. Ai, Y.L. Liu, L.H. Lu, J. Am. Chem. Soc. 131 (2009) 9496-9497. [18] L. Li, B. X. Li, D. Cheng, L.H. Mao, Food Chem. 122 (2010) 895-900. [19] W. Chen, H.H. Deng, L. Hong, Z.Q. Wu, S. Wang, A.L. Liu, X.H. Lin, X.H. Xia, Analyst 137 (2012) 5382-5386. [20] N. Kumar, R. Seth, H. Kumar, Anal. Biochem. 456 (2014) 43-49. [21] Z.M. Wang, D.H. Chen, X. Gao, Z.H. Song, J. Agr. Food Chem. 57 (2009) 3464-3469. [22] H.J. Zeng, R. Yang, Q.W. Wang, J.J. Li, L.B. Qu, Food Chem. 127 (2011) 842-846. [23] J.J. Zhang, M. Wu, D.H. Chen, Z.H. Song, J. Food. Compos. Anal. 24 (2011) 1038-1042. [24] X.S. Tang, X.Y. Shi, Y.H. Tang, Z.J. Yue, Q.Q. He, Luminescence 27 (2012) 229-233. [25] J.L. Manzoorj, M. Amjadi, J. Hassanzadeh, Microchim. Acta 175 (2011) 47-54. [26] K.C. Grabar, R.G. Freeman, M.B. Hommer, M.J. Natan, Anal. Chem. 67 (1995) 735-743. [27] S.L. Cumberland, G. F. Strouse, Langmuir 18 (2002) 269-276. [28] J.X. Du, Y.D. Wang, W.M. Zhang, Chem. Eur. J. 18 (2012) 8540-8546. [29] M.S. Filigenzi, B. Puschner, L.S. Aston, R.H. Poppenga, J. Agr. Food Chem. 56 (2008) 7593-7599.

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Figure captions Fig. 1. Ultraviolet-visible absorption spectra of (b) 1.92 nmol/L AuNPs solution and (c) the mixed solution of 1.92 nmol/L AuNPs and 1.6×10-5 mol/L melamine, and (a) fluorescence spectrum of 4.0 µmol/L fluorescein solution. Fig. 2. Fluorescence spectra of (a) 4.0 µmol/L fluorescein solution, (b) the mixed solution of 4.0 µmol/L fluorescein and 1.92 nmol/L AuNPs, and (c) the mixed solution of 4.0 µmol/L fluorescein with 1.92 nmol/L AuNPs and 1.6×10 -5 mol/L melamine. Fig. 3. TEM images of (A) AuNPs solution and (B) the mixture of AuNPs with melamine. Fig. 4. Effect of AuNPs concentration on the restored CL intensity. Conditions: 0.12 mol/L H2O2, 60.0 µmol/L TCPO, 4.0 µmol/L fluorescein (in 0.05 mol/L tris-HCl buffer, pH 7.5), 10 min of incubation time, and 3.2×10-9 mol/L melamine. Fig. 5. Effect of the incubation time on the restored CL intensity. Conditions: 0.12 mol/L H2O2, 60.0 µmol/L TCPO, 4.0 µmol/L fluorescein (in 0.05 mol/L tris-HCl buffer, pH 7.5), 1.92 nmol/L AuNPs, and 3.2×10 -9 mol/L melamine. Fig. 6. Selectivity of AuNPs-based CRET for responding to 3.2×10 -9 mol/L melamine, 3.2×10-8 mol/L Ca2+, Mg2+, Zn2+, SO42-, glucose, lactose, 3.2×10-7 mol/L K+, Na+, Fe3+, Cl-, NO3-, and 1.6×10 -6 mol/L sucrose. Conditions: 0.12 mol/L H2O2, 60.0 µmol/L TCPO, 4.0 µmol/L fluorescein (in 0.05 mol/L tris-HCl buffer, pH 7.5), 1.92 nmol/L AuNPs, and 10 min of incubation time.

13

Table 1 A comparison of various CL methods for the determination of melamine CL systems used

Linear range

Detection

Reference

limit Luminol–myoglobin

reaction, 0.01–50 ng/mL

3 pg/mL

21

0.2–80 µg/mL

0.12 µg/mL

22

2.5–250 pg/mL

0.9 pg/mL

23

9.0-7000.0 ng/mL

3.5 ng/mL

24

0.01–35 ng/mL

8 pg/mL

25

TCPO–H2O2–fluorescein-AuNPs

3.2×10-12–3.2×10 -7

3×10-13

This

reaction, CRET

mol/L

mol/L

method

inhibitory Luminol–H2O2 reaction, enhancing

Lumiol-K3Fe(CN)6 reaction, enhancing Permanganate–formaldehyde–Au/ Ag NPs reaction, inhibitory

14

Table 2 Determination of melamine in spiked milk samples Samples

Added (10-8 mol/L)

Found(10-8 mol/L)

1

0.0

0.0

0.9

2

3

4

Recovery (%)

RSD(%, n=5)

0.85

94.4

1.8

3.00

3.06

102.0

4.4

6.00

6.15

102.5

4.5

9.00

9.38

104.2

1.7

0.0

0.0

0.9

0.89

98.9

1.5

3.00

3.11

103.7

2.7

6.00

5.93

98.8

2.6

9.00

8.47

94.1

2.7

0.0

0.0

0.9

0.92

102.2

1.7

3.00

2.87

95.7

2.3

6.00

6.22

103.7

2.8

9.00

8.79

97.7

2.4

0.0

0.0

0.9

0.87

96.7

1.5

3.00

3.08

102.7

2.7

6.00

5.73

95.5

2.3

16

9.00

8.82

98.0

17

2.9

18

Highlights


A chemiluminescence resonance energy transfer method was developed for melamine.
Melamine modulates the dispersed and aggregated state of AuNPs.
The assay is simple, sensitive and specific for melamine.

19