Author's Accepted Manuscript

An upconversion fluorescence resonance energy transfer nanosensor for one step detection of melamine in raw milk Qiongqiong Wu, Qian Long, Haitao Li, Youyu Zhang, Shouzhuo Yao

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PII: DOI: Reference:

S0039-9140(15)00010-7 http://dx.doi.org/10.1016/j.talanta.2015.01.005 TAL15330

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Talanta

Received date: 31 October 2014 Revised date: 2 January 2015 Accepted date: 5 January 2015 Cite this article as: Qiongqiong Wu, Qian Long, Haitao Li, Youyu Zhang, Shouzhuo Yao, An upconversion fluorescence resonance energy transfer nanosensor for one step detection of melamine in raw milk, Talanta, http://dx. doi.org/10.1016/j.talanta.2015.01.005 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.




An Upconversion Fluorescence Resonance Energy transfer Nanosensor for One Step Detection of Melamine in Raw Milk Qiongqiong Wu, Qian Long, Haitao Li, Youyu Zhang*, Shouzhuo Yao Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research (Ministry of Education), College of Chemistry and Chemical Engineering, Hunan Normal University, Changsha 410081, PR China


Abstract Here we report a nanosensor based on fluorescence resonance energy transfer (FRET) between upconversion nanoparticles (UCNPs) and gold nanoparticles (AuNPs) for melamine detection. The positively charged UCNPs as donor and the negatively charged AuNPs as acceptor bound together via electrostatic interaction, which caused the fluorescence quenching of UCNPs. Upon addition of melamine, AuNPs were released from the surface of UCNPs and aggregation due to the N-Au interaction between melamine and AuNPs, which results in the fluorescence of UCNPs gradually recovered. Under the optimal conditions including media pH (7.0), the concentration of AuNPs (1.23 nM) and incubation time (12 min), the fluorescence enhanced efficiency shows a linear response to the melamine concentration ranging from 32 to 500 nM with a detection limit of 18 nM. Compared with other fluorescence methods, the fluorimetric nanosensor shows high sensitivity of 0.9677, ease of operation and can be used for the determination of melamine in raw milk samples.

Key words: Upconversion nanoparticles; Au nanoparticles; Fluorescence resonance energy transfer; Melamine detection





  








  

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1. Introduction Melamine is an organic base and a trimer of cyanamide, with a 1,3,5-triazine skeleton, which is mainly used in the production of plastic engineering, paint, adhesive and food packaing materials [1, 2]. However, melamine has been illegally added to milk powder, pet foods, and animal feeds to artificially increase the apparent protein content because of its nitrogen content as high as 66% by mass [3]. Since excessive ingestion of melamine beyond the safety limit (2.5 ppm in the United States and European Union, 1 ppm for infant formula in China) could lead to urinary system damage, kidney stone and ultimately death [4, 5]. Thus, it is quite necessary to develop a simple and sensitive method for melamine detection. Currently, various analytical methods have been reported for melamine assay, such as near infrared spectroscopy [6, 7], gas chromatography (GC) [8], mass spectroscopy (MS) [9], gas chromatography-mass spectrometry (GC-MS) [10, 11], high-performance liquid chromatography (HPLC) [12, 13], enzyme-linked immunosorbent assay (ELISA) [14, 15], surface-enhanced raman scattering spectroscopy (SERS) [16, 17]. Though some of the above methods have high sensitivity and accuracy, most of these are time-consuming and depend on professional technology, expensive instruments, and tedious sample pretreatment. Fluorescent methods have attracted great interests due to simple instruments and easy operations. Most of fluorescent methods for melamine detection mainly employ organic dyes compounds [18, 19]. However, these currently used organic fluorophores and dyes are vulnerable to chemical and metabolic degradation and easily photobleached [20]. In addition, organic dyes often have narrow absorption and broad emission spectra with long tailing, and the excitation and emission wavelengths of them are not sufficiently stable and could easily change with the ambient environment (pH, temperature) [21]. All of these characteristics limit their application. To circumvent these restriction, many quantum dots (QDs) [3, 22], have been used as fluorescent probe to detect melamine because of their bright photoluminescence, large Stokes shift, narrow emission and broad excitation [23, 24]. Nonetheless, their inherent toxicity and chemical instability limit the application in the assay of biomolecules [25]. Moreover, both organic dyes and semiconductor quantum dots are downconversion fluorescent materials which require excitation by short-wavelength ultraviolet (UV) light. The irradiation of hort-wavelength light may result in possible damage and the interference of protein fluorescense from biologic samples [26]. Thus, a simple, sensitive and suitable for complex samples approach is highly conceivable and needs further explorations. Since the eighties of the 20th century, rare-earth (RE) doped upconversion nanoparticles (UCNPs) emiting higher-energy visible light when excited by low-energy NIR light have recently aroused considerable attention [27]. Compared with conventional fluorescent labels, the unique luminescence mechanism of UCNPs possess several advantages such as (1) improved detection sensitivity owing to no autofluorescence, (2) the minimum photodamage to living organisms due to deeper NIR light penetration. (3) good chemical and physical stability, and low toxicity [28, 29]. On the basis of the advantages of UCNPs, a simple efficient fluorescence resonance energy transfer (FRET) system between positively charged UCNPs and negatively charged gold nanoparticles (AuNPs) was &






constructed to develop a nanosensor for melamine detection. As shown in Scheme 1, melamine could cause the aggregation of AuNPs by N-Au interaction, which influence the FRET system, and the fluorescence of UCNPs recovered. This method has been successfully applied to the determination of melamine in milk samples with satisfactory recovery from 98.8 to 102%.


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  The size and morphology of UCNPs and AuNPs were characterized by transmission electron microscopy (TEM) images using a JEOL-1230 TEM (JEOL, Japan). The fluorescence spectra were measured using an F4500 fluorescence spectrophotometer (Hitachi Ltd, Japan) with an external 980 nm laser diode (Hi-Tech Optoelectronic Co., Ltd. China) as the excitation source. Fourier transform infrared spectra (FT-IR) in the wavenumber range of 4000 to 400 cm-1 were recorded on a Nicolet Nexus 670 FT-IR spectroscope (Nicolet Instrument Co., USA). The crystalline phases of UCNPs were characterized using a Rigaku 2500 (Japan) Xray diffractometer (XRD). The absorption spectra were collected on an UV-245 spectrophotometer (Shimadzu Co., Japan). A Nano-ZS Zetzsozer ZEN3600 (Malvern Instruments Ltd., U.K.) was used to measure the Zeta potential of UCNPs and AuNPs.



   Rare-earth oxides used in this work, including yttrium oxide (Y2O3), ytterbium oxide (Yb2O3) and erbium oxide (Er2O3), were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and dissolved in hot nitric acid and then dissolved in deionized water to achieve final concentrations of 0.4 M, 0.2 M, 0.05 M, respectively. Chloroauric acid (HAuCl4), hexadecyl trimethyl ammonium bromide

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CTAB , melamine

were obtained from Sigma (Shanghai, China). The buffer solutions with different pH were prepared by 0.01 M KH2PO4-Na HPO4. All other chemicals (99%, Merck) used in this work were of analytical grade and without further purification, and Millipore Milli-Q ultrapure water (Millipore, • 18 MŸ cm) was used throughout the experiments.


  








The UCNPs of NaYF4:Yb3+, Er3+ were synthesized according to the previously reported method [30, 31]. Briefly, 0.2925 g ethylenediaminetetraacetic acid disodium salt (EDTA) was added to the solution containing 1.315 mL of 0.4 M Y(NO3)3, 0.105 mL of 0.2 M Yb(NO3)3 and 0.105 mL of 0.05 M Er(NO3)3 under stirring and the pH was adjusted to 8.0, then 10 mL glycol and 0.0675 g CTAB were added to the solution, after the solution became clear under ultrasonic stirring, another aqueous solution containing 0.5 mL hydrofluoric acid (HF) was added dropwise to the above solution with vigorous stirring for 0.5 h. Finally, the mixture was transferred into a teflon-lined autoclave and heated to 180 °C for 18 h. Then, the solution was cooled to room temperature. The nanocrystals were precipitated from the solution by centrifugation. The precipitates was washed with deionized water at first and than washed with ethanol. This washing procedure was repeated for three times. The product was dried under vacuum before it was to be used.


     AuNPs were prepared by the citrate reduction of HAuCl4 using the method described in the previous literature [32]. Typically, 100 mL chloroauric acid (HAuCl4) solution (containing 0.5 mL 2% HAuCl4) was firstly heated to boiling, and then 1.8 mL 1% sodium citrate solution was rapidly added to the boiled HAuCl4 solution under vigorous stirring. The mixed solution was boiled for 10 min and further stirred without heating for another 15 min. The color of the solution changed from pale yellow to wine-red and the solution was cooled to room temperature and then stored in the refrigerator (4 oC) for further use.



     





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“turn-on” quantitative assays. As described in Fig. 3A, the maximum quenching is observed when 1.23 nM AuNPs was added into the UCNPs solution. To avoid the interfering of excess AuNPs, 1.23 nM was used in the following experiments. In this study, the fluorescence enhanced efficiency is used as a standard for choosing the best condition. Fluorescence enhanced efficiency is defined as (F-F0)/F0, and F and F0 represent the fluorescence intensity in the presence and absence of melamine. We also investigated the time dependence of the reaction between the UCNPs-AuNPs system and melamine. As shown in Fig. 5A, the enhanced fluorescence efficiency remained stable when the incubation time reached 12 min, suggesting that the reaction of melamine and AuNPs reached equilibrium. To ensure maximal fluorescence recovery and obtain stable signal, 12 min was employed as the incubation time. Finally, the effect of pH on the fluorescence enhanced efficiency was investigated in the range from pH 4.0 to 10.0. As shown in Fig. 5B, the maximal fluorescence enhancement is obtained in pH 7.0 solution. Therefore, a 0.01 M KH2PO4-Na2HPO4 buffer solution with pH 7.0 was used throughout in this study.


    



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*Graphical Abstract (for review)

Highlight

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 2 3 4 5 6 7

Scheme 1 Schematic illustration of the "turn-on" fluorescence assay for detection of melamine.

16

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1 2 3

Fig. 1 Typical TEM images of UCNPs (A), AuNPs (B), UCNPs and AuNPs (C) , and UCNPs and AuNPs in the presence of melamine (D).

17

1.2

1.2

1.0 0.8

a b

1.0 0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0

0.0

A

Normalized Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

400 450 500 550 600 650 700 750 1

Wavelength (nm)

2

Fig. 2 spectral overlap: normalized fluorescence spectrum (λexc = 980 nm) of UCNPs (curve a) and

3

absorption spectrum of AuNPs (curve b).

4

18

3 4 5

A

1.0 0.8 0.6 0.4 0.2 0.0

6 450

7

4.5 B 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 I0/I

2

1.2

I0/I

1 FL Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

500

550

600

650

700

2.0 1.8 1.6 1.4 1.2 1.0 0.2

0.3

0.4

0.5

0.6

0.7

0.8

cAuNPs (nM)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

750

cAuNP (nM)

Wavelength (nm)

8 9

Fig. 3 Fluorescence emission spectra of UCNPs along with the concentrations of AuNPs (A). The

10

linear fitting of the fluorescence response vs. AuNPs concentration (B). I0 and I stand for the

11

fluorescent intensities of UCNPs in the absence and presence of AuNPs, respectively. The

12

concentration of UCNPs is 0.06 mg/mL.

13

19

Normalized Intensity (a.u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1.2 a

1.0

b

0.8

d

0.6 c

0.4 0.2 0.0 450

500

550

600

650

700

750

Wavelength (nm)

1 2

Fig. 4 Fluorescence spectra of UCNPs (a); and UCNPs and melamine (b); UCNPs and AuNPs (c);

3

and UCNPs, AuNPs and melamine (d). [UCNPs]: 0.06 mg/mL, [AuNPs]: 1.23 nM, [melamine]:

4

0.5 mM.

20

1 2 0.6

3

5 6 7 8

B

0.40

0.4

(F-F0)/F0

4

0.45

A

0.5

(F-F0)/F0

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

0.3 0.2 0.1

0.35 0.30 0.25

0.0

0.20 0

2

4

6

8

10

12

14

3

Time (min)

4

5

6

7

8

9

10

pH

9 10 11

Fig. 5 Effects of incubation time (A) and media pH (B) on the fluorescence responses of UCNPs-AuNPs nanosensor for melamine detection.

12

21

11

1

1.0 0.8

1.0 0.9 0.8 0.7 0.6 0.5

0.6

0.0

0.1

0.2

0.3

0.4

0.5

cmelamine (mM)

0.4 0.2 0.0 450

2

Normalized intensity

1.2

Normalized intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

500

550

600

650

700

750

Wavelength (nm)

3

Fig. 6 Fluorescence spectra of UCNPs-AuNPs nanosensor in the presence of different

4

concentration of melamine. The concentration of melamine (from down to up) is 0, 0.032, 0.08,

5

0.12, 0.25, 0.35, 0.4, 0.5 mM, respectively. [AuNPs]: 1.23 nM. [UCNPs]: 0.06 mg/mL. pH:7.0.

6

Inset: the enhanced fluorescence intensities as a function of the concentration of melamine.

22

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65

1

2 3

Fig. 7 Fluorescence enhanced efficiency of UCNPs-AuNPs nanosensor upon the addition of

4

melamine and other substances: (1) blank; (2) lactose (16mM); (3) glucose (16mM); (4) Mg2+

5

(16mM); (5) Fe2+ (16mM); (6) Zn2+ (16mM); (7) Fe3+ (16mM); (8) Na+ (16mM); (9) Mn2+ (16mM);

6

(10) Cl- (16mM); (11) SO42- (16mM); (12) NO3- (16mM); (13) lysine (0.35mM); (14) cysteine

7

(0.35mM); (15) histidine (0.35mM); (16) melamine (0.35mM).

23