A Rapid Colorimetric Detection of Melaminein Raw Milk by Unmodified Gold Nanoparticles

Hai-bo Xing & Yuan-gen Wu & Shen-shan Zhan &

Pei Zhou

Received: 13 September 2012 /Accepted: 1 January 2013# Springer Science+Business Media New York 2013

Abstract We report a rapid, visual, facile colorimetric de-tection of melamine in raw milk by unmodified gold nano-particles (AuNPs). AuNPs can be induced by melamine fromdispersion to aggregation rapidly, along with the color changefrom red to blue (or purple). After the optimization of reactionconditions, the whole analysis only needs about 20min includ-ing the simple pretreatment we designed. The analysis can beobserved by the naked eye and simple to operate withoutsophisticated instruments. In the raw milk, the present limitof detection for melamine is down to ∼70 ppb. Furthermore,we explore the interaction principle between melamine andcitrate-stabilized AuNPs in this work and establish the foun-dation for modifying the AuNPs and optimizing the system ofAuNPs–melamine in the future.

Keywords Melamine . Colorimetric detection . Rawmilk .

Unmodified gold nanoparticles . Interaction principle

Introduction

Melamine (1,3,5-triazine-2,4,6-triamine) is a industrial chem-ical compound which is widely used in plastics, fertilizer,melamino-formaldehyde resin, paper, and other products(Yang et al. 2009). For its high nitrogen content (66 % bymass), it is illegality added into food, feed, and even milk inorder to increase the apparent protein content based on the totalnitrogen, which is measured by the conventional standardKjeldahl and Dumas tests (Serdiuk et al. 2010). Recently,melamine is considered to have low toxicity and the LD50 ofrat given with oral administration is greater than 3 g/kg. Smallamounts of occasional intake are nontoxic for the human body,but long ingestion of melamine may cause reproductive andurinary system damage, leading to bladder and kidney stonesand inducing bladder cancer (Cao et al. 2009). In 2008, inChina, high levels of melamine were found in milk and otherdairy products, causing kidney failure and stones in tenthousands of infants. In 2007, in USA, protein feeds for petsmixed with melamine killed lots of cats and dogs (Choi et al.2010). Therefore, in 2008, The World Health Organization'sfood safety experts decided that the amount of melamine aperson could stand per day, the “tolerable daily intake,” was0.2mg/kg of bodymass (Wu et al. 2012a). In the USA and EU,the ingestion of melamine level above the safety limit is2.5 ppm, and in China, it is 1 ppm. So, it is important to setup a rapid, effective, and sensitive detection of melamine.

Nowadays, the main methods to detect melamine in milkand dairy products are the following: high-performance liquidchromatography (HPLC) (Peng et al. 2011), HPLC–massspectrometry, gas chromatography, ultra performance liquidchromatography-mass spectrometry/mass spectrometry, andcapillary zone electrophoresis (Li et al. 2010). Additionally,there are also some new methods to monitor melamine, such assurface enhanced Raman spectroscopy, fluorescence, spectro-photometric absorption, and chemiluminescence (Ding et al.2010). Although these methods are highly sensitive, most of

H.-b. Xing :Y.-g. Wu : S.-s. Zhan : P. ZhouSchool of Environmental Science and Engineering, Shanghai JiaoTong University, Shanghai, China

H.-b. Xinge-mail: michaelyanzi@163.com

H.-b. Xing :Y.-g. Wu : S.-s. Zhan : P. ZhouKey Laboratory of Urban Agriculture (South),Ministry of Agriculture, Shanghai, China

P. Zhou (*)School of Agriculture and Biology, Shanghai Jiao Tong University,Shanghai, 200240, Chinae-mail: peizhousjtu@163.com

P. ZhouBor S. Lug Food Safety Research Center, Shanghai Jiao TongUniversity, Shanghai, China

H.-b. Xing : P. ZhouThe Center for Food Nutrition and Safety, Shanghai Jiao TongUniversity, Shanghai, China

Food Anal. MethodsDOI 10.1007/s12161-013-9562-3

them are either time-consuming due to exhaustive pretreatmentor costly due to the expensive instrumentation. Therefore, thesimple, rapid, low-cost, easy operated methods are required todevelop for detecting melamine in milk and dairy products.

It is well known that gold nanoparticles (AuNPs) exhibitvisual sensing properties; the use of AuNPs as a colorimetricsignal depends on their colors of red or blue corresponding totheir dispersion or aggregation (Wu et al. 2011, 2012b).According to this, several colorimetric systems have been setup to detect proteins, ions, viruses, DNA, cancerous cells, β-lactamase, and, of course, melamine. For detecting melamine,the surface-functionalized AuNPs together with analyte cantransform from dispersive to aggregated; meanwhile, the colorof AuNPs changes (Guo et al. 2010). One research reports thatthe color change induced by the triple hydrogen bonding recog-nition between melamine and a CA derivative grafted on thesurface of gold nanoparticles can be used for reliable detectionof melamine (Han and Li 2010; Lee et al. 2010; Qi et al. 2010).In some assays, electrostatic interactions between melamineand citrate-stabilized AuNPs have been developed for thedetection. Actually, it is not only because of electrostaticinteraction that AuNPs can thus be cross-linked directly inthe presence of certain amounts of melamine (Araujo et al.2012; Campbell et al. 2007; Roy et al. 2011). So, in this work,the structure and reaction principle have been discussed be-tween melamine and unmodified AuNPs. Moreover, based onthis, we optimized this rapid visual detection of melamine byunmodified gold nanoparticles and hope to provide the basicfor modifying the AuNPs or linking with varied analytes inorder to improve the sensitivity for detection.

Experimental

Materials and Apparatus

Chloroauric acid, sodium citrate, and sodium chloride werebought from Shanghai Chemical Reagent Company(Shanghai, China). Melamine was obtained from Sigma-Aldrich (Milwaukee, WI, USA). The raw milk was purchasedfrom a local cattle farm nearby. All reagents were of analyticalgrade. Milli-Q water of 18MΩcm was used in all experiment.Absorption spectra were performed on a UV-2410 PC UV–Vis Spectrophotometer (SHIMADZU) and a Bio-Tek EpochMicroplate Reader (Bio-Tek Instrument, USA). The pHmeas-urements were recorded on DELTA 320 pH meter(METTLER TOLEDO).

Preparation of AuNPs

Following usage, all glassware was cleaned in aqua regia-prepared 3:1 HNO3/HCl, washed thoroughly in water, anddried in the dry oven. AuNPswith a diameter of 18.0 nmwere

prepared by the trisodium citrate reduction method (Li et al.2010). One hundred milliliters of 0.01 % (w/w) HAuCl4 sol-utions was heated until boiling. After 2 min, 3.5 mL of 1 %sodium citrate solution was added quickly with vigorous stir-ring. The color immediately changed from pale yellow topurple, then to dark red, and to bright red in the end. Afterabout 15 min, the heating was stopped. The solutions werestirred continuously about 15 min until it cooled to roomtemperature. Finally, the cooled solution was diluted with100 mL of water and stored in the refrigerator at 4 °C.According to this procedure, we obtained nanogolds withconsistent particle size of 18 nm.

Principle of Detection of Melamine Using AuNPs

A series of analysis were performed to assess the interactionbetween the AuNPs and melamine as follows: First, standardsolution of melamine was diluted into different concentration,mixed with AuNPs as 1:1, and the final concentrations ofmelamine were 0, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, and 10 μM(1.0 μM=0.126 ppm). The samples were sent to theInstrumental Analysis Center of SJTU to find the melamineconcentration-dependent size increase in gold–melamine par-ticle aggregation by dynamic light scattering (DLS) analysis.Second, to understand the melamine aggregation mechanism,we chose six different amino compounds (1.0 μM) mixedwith AuNPs (Chi et al. 2010). With pure water and melamineas the negative control and positive control, we balancedmixed sample and AuNPs for 5 and 300 min at room temper-ature then quantified by the absorption ratio (A640/A520).Third, AuNPs were premixed with NaCl of different concen-trations (0, 10.0, and 20.0 mM) to detect melamine in 5 min.

Colorimetric Detection of Melamine

A typical colorimetric analysis was realized as follows: First, aseries concentration of melamine were mixed with AuNPs as1:1, and the final concentrations of melamine were 0, 0.4, 0.6,0.8, 1.0, 2.0, 4.0, and 10μM.After reaction for 5 min, the colorchange was observed by both the naked eyes and the spectro-photometer scanned from 400 to 800 nm. Second, the mixturemixed the same way as above was observed by both nakedeyes and the spectrophotometer scanned at 640 and 520 nmafter reaction for 0, 5, 10, 30, 60, 120, 300, and 600min. Third,the mixture of melamine (1.0 μM) and AuNPs was adjustedfrom pH1 to pH14 and scanned by the spectrophotometer at640 and 520 nm after reaction for 10 min.

Developing of Method

A series concentration of melamine were first added into rawmilk samples, and after pretreatment, they were mixed withAuNPs as 1:1, and the final concentrations of melamine were

Food Anal. Methods

0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4,1.5, 1.6, 1.7, 1.8, 1.9, and 2.0 μM. The samples were scannedby the spectrophotometer at 640 and 520 nm after reaction for10 min. After the standard curve was finished, the fortifiedrecovery test of melamine in raw milk was taken based on it.

Result and Discussion

Principle of Melamine Detection Using Unmodified AuNPsas Colorimetric Probe

Figure 1 illustrated that melamine has a strong interactionwith AuNPs. When melamine exists, this interaction willreduce the stability of melamine against aggregation andcause AuNPs' visible color changes from red to blue infew seconds. In addition, AuNP solution shows a particularcolor because of the surface electron oscillations induced byvisible light of suitable wavelength, which is dependent oninterparticle spacing (Ai et al. 2009). According to the threeamine groups (–NH2) of melamine, it seems that AuNPs areeasily bind to amine functional groups.

The principle of DLS to measure the size of AuNPs wasbased on the Brownian motion of particles that cause aDoppler shift of incident laser light. In our research, thismethod was performed to assess the interaction between themelamine and AuNPs (Wei et al. 2010). The sizes followedwere based on the average of three repeated measurements.Figure 2 showed a relationship between melamine concen-tration and size increase in AuNPs–melamine particle ag-gregation. The color changes from red to blue happened inmelamine concentration higher than 0.6 μM, as the averagehydrodynamic particle diameter of AuNPs–melamine waslarger than 60 nm. There were reports that aggregate sizeand interparticle distances caused this surface plasmonresonance-based colorimetric shift. So, with the melamine

added more in AuNPs, the mixture color will change fromred to blue, and the average particles of melamine–AuNPswill become greater, just as Fig. 2 reveals.

We compared the colorimetric difference of AuNPs to sixamino compounds, 1—ammonium hydroxide, 2—propyl-amine, 3—1,2-cyclohexanediamine (Wei et al. 2010), 4—poly-L-lysine (Liang et al. 2011), 5—adenine, 6—triazine,and 7—melamine, to deeply find out the melamine-AuNPsaggregation mechanism. For 5 min, there was no visiblecolor change of AuNPs with the addition of other six com-pounds. Testing by the UV–Vis spectroscopic response ofAuNPs with the six compounds of 1 μM in solution (Fig. 3),the A640/A520 ratios for 1 and 2 are the same as the blank.Meanwhile, the A640/A520 ratios for 3, 4, 5, and 6 are a littlehigher than the blank, which means that 3, 4, 5, and 6change the interparticle distances or cause the aggregationof melamine, but not obvious. For 300 min, there was stillno visible color change of AuNPs with 1 and 2, but the colorof AuNPs with amino compounds 3, 4, 5, and 6 changedfrom red to purple when the blank was red and 7 was deepblue. The A640/A520 ratios of 3, 4, 5, and 6 for 300 minfurther prove that they can cause AuNP aggregation, but notas much as melamine. There are no effects to AuNPs with 1and 2. In conclusion, the exocyclic amino groups induce theaggregation of AuNPs; the ring nitrogen atoms and thesymmetrical structure of compounds will make this aggre-gation obvious.

As we mentioned above, the interaction between mela-mine and AuNPs led to the aggregation of AuNPs with thecolorimetric signal change. Wei et al. (2010) pointed outearlier that the sensitivity and dynamic range of the colori-metric signal are dependent on the resistance of AuNPs toaggregation and could be adjusted by changing the buffercomposition of the AuNPs. According to this, we chosesodium chloride to investigate this inference because sodiumchloride is famous to destabilize the AuNPs. AuNPs were

Fig. 1 Schematicrepresentation of thecolorimetric detection formelamine

Food Anal. Methods

premixed with NaCl of 0, 10.0, and 20.0 mM to detect mela-mine (Fig. 4). As Fig. 4 illustrated, the presence of NaCl in thissystem of melamine–AuNPs obviously amplified the signalsof sensitivity and dynamic range. If we can figure out thecorrect relationship between the addition of NaCl and thechange of melamine–AuNP signals, we can pretreat theAuNPs so that a significant color change can happen aroundthe safety level when we want to determine whether there ismelamine above the safety level in the samples.

Colorimetric Detection of Melamine

UV–Vis Absolution Spectra of AuNPs As reported previously,melamine has a strong electrostatic interaction with citrate-stabilized AuNPs, which decrease the stability of AuNPsand cause the visible color changes (Chi et al. 2010). Thereare still problems using this character as a probe to indicate the

existence of melamine. Figure 5 shows the color changes andUV spectra of melamine–AuNPs after reaction for 5 min. Theblank group shows that citrate-stabilized AuNPs are red be-cause of their surface plasma resonance at 520 nm. Alongwith the increase of melamine added in the mixture ofAuNPs–melamine, the solution color changes from winered, purple to blue progressively, and the surface plasmaresonance at 640 nm increases; on the contrary, thesurface plasma resonance at 520 nm decreases. For5 min, clear color changes were observed to purple, blue at amelamine final concentration 2.0 μM. Meanwhile, the aggre-gation of AuNPs can be monitored by UV–Vis spectroscopyat a melamine final concentration of 0.8 μM. From Fig. 5, wecan see that more melamine will cause more aggregation ofAuNPs and the significant aggregation of nano particles in thepresence of more than 2.0 μM (252 ppb) melamine for reac-tion 5 min.

Fig. 3 The A640/A520 ratios of AuNPs with the addition of seven aminocompounds with different concentration

Fig. 2 DLS demonstration of melamine concentration-dependentincreases in particle size

Fig. 5 Visual color change and the spectrophotometric demonstration

Fig. 4 The absorbance ratio of AuNPs–melamine in the presence ofNaCl

Food Anal. Methods

Time Specificity About Aggregation of AuNPs We also choseeight groups to examine the aggregation of AuNPs in thepresence of melamine for 0∼600 min in order to evaluate theaggregation kinetics of AuNPs. As shown in Fig. 6, thehigher the concentration of melamine is, the faster theAuNPs aggregate. When there is a high melamine concen-tration (>1.0 μM), the extinct ratio rises fast in the initialstage. At the concentration of 1.0 μM, the absorption ratio(A640/A520) begins to increase from original 0.108 to 0.118obviously during the first 10 min with the mixture colorchanged from wine red to light purple. At the high concen-tration (>1.0 μM), the ratio increases more obviously duringthe first 10 min, with the color changed from wine red todark blue. At the concentration of 0.6 and 0.8 μM, the ratiobegins to increase during 10 to 30 min, and the mixturecolor will become light blue, not dark blue at last. It meansnot all the particles of AuNPs get aggregated at this concen-tration of melamine. At the concentrations of 4.0 and

10.0 μM, the ratio decreases after reaction for 120 min. It isbecause all the particles of AuNPs get aggregated and depos-ited, and the solution color becomes clear from dark blue. Inconclusion, it is enough for us to detect the low concentrationof melamine (<1.0 μM) by absorption ratio after reaction for10 min. We do not need more obvious result (after more than30 min) because rapidity is also important.

The Optimization of pH The effects of pH can be serious tothe interaction of AuNPs with small molecule. As we know,melamine is alkalescence with the pKa of 5.05 (Liang et al.2011); the pH of solution also affects the solubility ofmelamine. We fix the melamine concentration at 1.0 μMand adjust the acetic acid buffer solution with hydrochloricacid and sodium hydroxide. Figure 7 shows that the pH ofsolution hardly affects the aggregation of AuNPs, and theabsorption ratio (A640/A520) is very low in strong base mediaand strong acidic media (pH>12.0, pH<2.0). It is becausemelamine can be transformed into cyanuric acid losing threeamino groups in strong basic media and strong acidic media

Fig. 6 The absorption ratio for different concentration of melamine indifferent time

Fig. 7 Effect of pH on the absorption ratio (A640/A520)

Fig. 8 The calibration curves of melamine in raw milk

Table 1 Application of this method and HPLC to detect the melaminein raw milk samples spiked with different amounts of melamine

Sample Concentration of melamine (ppm) Recovery (100 %)Added Found (AuNPs)

1 0.200 0.24 (±0.01) 120

2 0.500 0.46 (±0.02) 92

3 1.000 1.05 (±0.05) 105

4 2.000 1.98 (±0.10) 99

Sample Concentration of melamine (ppm) Recovery (100 %)Added Found (HPLC)

1 0.200 0.21 105

2 0.500 0.51 102

3 1.000 1.00 100

4 2.000 1.99 99.5

Food Anal. Methods

(Chi et al. 2010), and cyanuric acid cannot lead to theaggregation of AuNPs. At pH8.0, the absorption ratio isthe highest during the pH1.0 to 14.0, thus we chose 7.0 asthe best pH for the detection media.

The Calibration Curves of Melamine in Raw Milk To dem-onstrate whether the citrate-stabilized AuNPs can be usedfor the detection of melamine in milk powder, we addeddifferent amounts of melamine to the raw milk before sam-ple pretreatment. Milk is a complex system and its variouscomponents serve as potentially interactive species withmelamine regardless of detection methodology. For thecasein-based milk, the casein exists in milk with the formof water-soluble calcium salt of a phosphoprotein; acidtreatment removes the calcium cation, leaving a water-insoluble phosphoprotein (Li et al. 2010). So, we add aceticacid into the milk and centrifugate the samples to separatethe liquid component from the white, opaque precipitation.Then, we adjust pH to 8.0 and supply the solution volumewith pure water.

When the concentration of melamine is high (>2.0 μM),the absorption ratio increases too fast for us to control andthe process of color shift is hard to observe clearly in thefirst 10 min. So, we chose the concentration range of0∼2.0 μM to evaluate the melamine concentration by com-paring the A640/A520 value. Beyond the concentration range,the samples can be diluted into this range probably anddetected. As shown in Fig. 8, melamine in the raw milksamples could be quantified within the range of 0.6 to2.0 μM, and the detection limit is less than 0.6 μM (thefinal concentration). Since ingestion of melamine at levelsabove the safety limits are 2.5 ppm (20 μM) in USA and EUand 1 ppm (8 μM) for infant formula in China, thesecalibration curves of melamine can be used to detect themelamine in raw milk samples.

Detection of Melamine in the Raw Milk Samples Before sam-ple pretreatment, we added different amounts (0.2, 0.5, 1.0,and 2.0 ppm) of melamine to the raw milk. This proposedmethod and HPLC were applied to analyze melamine in theraw milk samples. As it shown in Table 1, the recoveries arebetween 90 and 120 % by AuNPs, while the recoveries arebetween 99 to 105 % by HPLC. It indicates that this methodusing AuNPs as a probe for rapid detection of melamine inraw milk is feasible.

Conclusions

In summary, a simple and rapid colorimetric assay for mela-mine has been demonstrated. Due to the interaction betweenAuNPs and melamine, the AuNPs can be induced from

dispersion to aggregation, meanwhile the color changes fromred to blue. This proposed detection of melamine can be takenafter simple pretreatment and the detection limit is far less thanthe safety limits in China. The whole analysis only needs about20 min including pretreatment, can be observed by the nakedeye, and simple to operate without sophisticated instruments.So, this cheap method will probably be used to detect mela-mine in other foods.What is more, in this work, we explore theinteraction principle between melamine and citrate-stabilizedAuNPs further and establish the foundation for modifying theAuNPs and optimizing the system of AuNPs–melamine in thefuture.

Acknowledgments This work was supported by the National High-Tech Research and Development Plan (2012AA101405), the SpecialFund for Agro-Scientific Research in the Public Interest of China(200903056), the National Natural Science Foundation of China(31071860), and the National key Technology R&D Program(2010BAK69B18).

Conflict of Interest There are no conflicts of interest.

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