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“Oxidative etching-aggregation” of silver nanoparticles by melamine and electron acceptors: An innovative route toward ultrasensitive and versatile functional colorimetric sensors
Analytica Chimica Acta 747 (2012) 92–98
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“Oxidative etching-aggregation” of silver nanoparticles by melamine and electron acceptors: An innovative route toward ultrasensitive and versatile functional colorimetric sensors Guang-Li Wang a,∗ , Xiao-Ying Zhu a , Huan-Jun Jiao a , Yu-Ming Dong a,b , Xiu-Ming Wu a , Zai-Jun Li a a b
The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009, PR China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
A novel concept for colorimetric sensing of MA based on non-crosslinking of AgNPs. The detection mechanism was based on “oxidative etching-aggregation” of unmodified AgNPs. This method was simple, selective and ultrasensitive. The detection limit for MA was as low as 0.08 nM when using H2 O2 as electron acceptor. The method also enabled the detection of H2 O2 with a detection limit of 0.2 nM.
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 11 August 2012 Accepted 16 August 2012 Available online 23 August 2012 Keywords: Colorimetric sensor Melamine Silver nanoparticles Oxidative etching-aggregation
a b s t r a c t An innovative and versatile functional colorimetric sensor for melamine (MA) and H2 O2 was developed with simplicity, excellent selectivity and ultrasensitivity. The detection mechanism was based on the “oxidative etching-aggregation” of silver nanoparticles (AgNPs) by the cooperation effect of MA and electron acceptors such as H2 O2 , ozone or Fe(NO3 )3 . The detection limits of this method for MA could reach as low as 0.08 nM, 0.16 nM and 3 nM when H2 O2 , ozone or Fe(NO3 )3 was used as an electron acceptor, respectively. When using H2 O2 as a typical electron acceptor, the method enabled the detection of H2 O2 with a detection limit of 0.2 nM. This proposed method offered a new way to design MA and H2 O2 sensors and might be easily extended to detect other nucleophilic reagents and electron acceptors based on colorimetric sensors. © 2012 Elsevier B.V. All rights reserved.
1. Introduction With the development of nanoscience and nanotechnology, more and more opportunities are provided for the application of nanomaterials in colorimetric sensing. Especially, gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) have emerged
∗ Corresponding author. Tel.: +86 510 85917090; fax: +86 510 85917763. E-mail address: [email protected] (G.-L. Wang). 0003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2012.08.019
as exciting materials as colorimetric probes because they have strong surface plasmon resonance and behavior that depends on the interparticle distance [1–4]. When the distances between the AuNPs/AgNPs become less than the average particle diameter, aggregation of the dispersed NPs occurred. Interparticle plasmon coupling of AuNPs/AgNPs occurred in aggregated NPs and the color of the NPs’ solution changed, which enabled the development of sensitive colorimetric sensors [5–7]. Recently, assays to detect melamine (MA) based on interparticle crosslinking aggregation by hydrogen-bonding or charge-transfer recognition between MA and
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surface modifiers of AuNPs were reported [8–18]. Although using surface modifiers to functionalize NPs is a successful tool to endow NPs with the recognition ability for MA, it makes the preparation of the NPs cumbersome and the detection procedure complicated. Moreover, surface modification usually deteriorates the superior optical properties of the metal NPs. Hence, it is desirable to develop an analytical method for the simple, sensitive and selective detection of MA without surface modification. It is worth noting that, for colorimetric assays, AgNPs have some advantages over AuNPs because they are cheaper and possess higher extinction coefficients compared to AuNPs with the same size [19]. In addition, AgNPs have reaction activeness though bulk Ag material is regarded as being inert. Herein, a novel concept for colorimetric detection of MA based on “oxidative etching-aggregation” of unmodified AgNPs with simplicity and ultrasensitivity was proposed. It was found that “oxidative etchingaggregation” of AgNPs could be very effective under the coexistence of MA with very low concentration (even at sub-nanomolar level) and electron acceptors. MA as a nucleophilic reagent could donate its electron pairs of N to the unoccupied orbital of AgNPs, which led to a more negative Fermi potential of AgNPs with excess electrons. Thus, AgNPs were more active toward oxidation by electron acceptors. In addition, MA could strongly complex with Ag+ produced by the oxidative etching of AgNPs through MA and electron acceptors. Chemisorption of the positively charged complex made of Ag+ and MA would lead to the decrease of the surface charges, resulting in non-crosslinking aggregation and significant change in color of the AgNPs. This method offered a new way to design MA sensors. More importantly, this work also provides possibilities to detect electron acceptors. Based on the above consideration, we demonstrated herein a simple and versatile functional colorimetric sensor for MA and H2 O2 with good selectivity and ultrasensitivity. This method opens up a new perspective for the development of highly sensitive colorimetric sensors based on the special reactivity of AgNPs with nucleophilic reagents and electron acceptors. 2. Experimental details 2.1. Chemical reagents Melamine, silver nitrate, trisodium citrate, hydrogen peroxide (30 wt%), sodium borohydride (96%), ferric nitrate were all obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade. All solutions were prepared with ultrapure water (18.2 M cm−1 ) obtained from a Healforce water purification system. 2.2. Apparatus UV–vis absorption spectra were carried out using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Photographs of the solutions were taken with Canon 530A digital camera. Resonance light scattering spectra were measured on a Cary Eclipse spectrofluorimeter (Varian, USA). The zeta potential and size distribution of the AgNPs were measured using a ZetaPALS zeta potential and particle size analyzer (Brookhaven, USA). High resolution transmission electron microscopy (HRTEM) images of AgNPs were obtained on a JEOL JEM-2100 transmission electron microscope (Hitachi, Japan). 2.3. Synthesis of Cit-AgNPs Citrate-capped AgNPs (Cit-AgNPs) were prepared by means of the chemical reduction of AgNO3 in the liquid phase according to the reported method [20] with modifications. To achieve this,
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20 mL of 1.0 mM AgNO3 was added to a 50 mL of solution containing 5.3 mM NaBH4 and 1.0 mM trisodium citrate. Then the mixture was allowed to react at room temperature for 18 h. 2.4. Colorimetric detection of MA and H2 O2 For MA sensing, electron acceptor (0.3 mL of 0.01 M H2 O2 or ozone (bubbled for 15 s) or 0.2 mL of 1 mM Fe(NO3 )3 ) was introduced to a solution containing 2 mL of Cit-AgNPs and certain amount of MA in a 5 mL calibrated test tube. Then the mixed solution was dissolved to volume with ultrapure water. The final concentrations were 0.6 mM, 0.42 mM and 0.04 mM for H2 O2 , ozone and Fe(NO3 )3 respectively. Twenty-five minutes later, the absorption spectra of the reaction mixture were recorded. Ozone was generated in a laboratory ozone generator using pure oxygen as gas source. The flow rate of oxygen was 15 mL min−1 , and the flow rate of ozone was 0.40 mg min−1 . To detect H2 O2 , different concentrations of H2 O2 were added to the solution containing 2 mL of Cit-AgNPs and 0.3 mL of 0.1 mM MA in a 5 mL calibrated test tube. Then the mixed solution was dissolved to volume with ultrapure water. The final concentration of MA was 6 M. 2.5. Colorimetric detection of MA and H2 O2 in real samples The method was used to detect MA in raw milk by standard addition method. The raw milk was pretreated as reported procedures [21]. The raw milk was purchased from local supermarkets. First, 1 mL of acetonitrile, 1 mL of CCl3 COOH, and 7 mL of water were added into 2.0 mL of raw milk. Then the mixture in a centrifuge tube was ultrasonically extracted for 20 min and then centrifugated at 5000 rpm for 20 min. The obtained supernatant was filtered two times. The obtained supernatant was adjusted to pH 8.0 with a small amount of 1 M NaOH. To quantify MA in raw milk, different amounts of MA were added to 2.0 mL of MA-free raw milk, then the mixture was handled as the steps described in the above sample preparation section. The obtained solutions were mixed with 2 mL of Cit-AgNPs before adding H2 O2 (0.3 mL, 0.01 M). To detect H2 O2 in rain water, a series of different concentrations of H2 O2 samples were prepared by “spiking” the rain water with different amounts of H2 O2 . The H2 O2 solution was added to a solution containing 2 mL of Cit-capped AgNPs and 0.3 mL of 0.1 mM MA. 3. Results and discussion 3.1. Colorimetric sensing mechanism The suspension of the as-synthesized Cit-AgNPs was yellow and transparent (Fig. S1), exhibited a characteristic and intense absorption peak around 400 nm owing to the surface plasmon excitation [22], which indicated a good dispersity of Cit-AgNPs in water. The negative charges of Cit on the surface of AgNPs acquired an electrostatic double layer, which provided a repulsive force between separated AgNPs and enabled the AgNPs to be dispersed stable in aqueous solution [23]. At first, Cit-AgNPs were directly utilized to quantify MA in aqueous solution. It was found that the color or the absorption of Cit-AgNPs did not change until the concentration of MA reached 10 M (Fig. S1). Through the coordinating interactions, nucleophilic primary amines as well as the ring nitrogens in hybrid aromatics of MA can bind onto the surface of metal NPs which are coordinatively unsaturated [24]. The adsorption of MA replaced the original weakly surface-bound citrate ions, neutralized the negative charge of Cit-AgNPs and induced aggregation of AgNPs. Particle aggregation led to color and absorption changes of
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the interval time between the addition of H2 O2 and MA into CitAgNPs. Considering that there was no reaction between MA and H2 O2 , a hypothesis was provided that intermediate species produced from the reaction of Cit-AgNPs and H2 O2 could interact with MA and played crucial role in the color change of Cit-AgNPs. Interestingly, it has been recently manifested that AgNPs catalyzed the decomposition of H2 O2 through a reformation process of AgNPs [27,28]. The reaction between AgNPs and H2 O2 generated superoxide anion and Ag+ (Eq. (1)). Ag+ could be quickly reduced to elementary Ag by superoxide anion and electron charged AgNPs (AgNP*− ) whose formation was mediated by the superoxide anion (Eq. (2)), resulting in the regeneration of AgNPs [27,28]. The redshift of the absorption band and slight absorption decrease of AgNPs after reaction with H2 O2 may be due to the particle size change of AgNPs resulted from the reformation process. Dynamic light scattering (DLS) result indicated that the average diameter of AgNPs increased from 74.8 nm to 76.5 nm after reaction with H2 O2 . •
AgNP + H2 O2 → Ag+ + O2− + 2H2 O •
AgNP + O2− → AgNP∗− + 2H2 O
Fig. 1. The photograph (top part) and evolution of absorption profiles (bottom part) of Cit-AgNPs in the presence of different concentrations of MA and 0.6 mM H2 O2 .
Cit-AgNPs [25]. Nevertheless, the color change induced by MA alone without any extra aid only occurred until the concentration of MA reached 10 M, which was higher than the safety limit (7.9 M for infant formula) defined by the UN food standard commission [26]. Exhilaratingly, upon the introduction of 0.6 mM H2 O2 into the mixture of Cit-AgNPs and MA, color change of Cit-AgNPs could be found even at 0.6 nM of MA (Fig. 1). To the best of our knowledge, this was the lowest concentration that could be detected with naked-eye using AuNPs or AgNPs (Table S1). However, it was found that the introduction of 0.6 mM H2 O2 alone could not induce any detectable color change of Cit-AgNPs (Fig. 2(b)). The absorption band of AgNPs red-shifted slightly from 397 nm to 400 nm and the absorption intensity weakened by a little (about 4.39%) after reaction with 0.6 mM H2 O2 (Fig. 2(b)). That is, the changes in color and absorption of Cit-AgNPs solution induced by the coexistence of H2 O2 and MA were much more obvious than the changes in color and absorption of Cit-AgNPs solution induced by H2 O2 or MA alone (Fig. 2b–d). Particle size and zeta potential measurements also confirmed the larger extent of changes in particle size and zeta potential of Cit-AgNPs in the coexistence of MA and H2 O2 than that in the presence of MA or H2 O2 alone (Fig. 2B), which was in accordance with the much more obvious color and absorption changes of Cit-AgNPs under the same experimental conditions. Hence, it should be pointed out that the color change in Fig. 1 should be ascribed to the cooperation effect of MA and H2 O2 . The adding ways of MA and H2 O2 to Cit-AgNPs solution also significantly affected the color change of Cit-AgNPs. When MA was added to Cit-AgNPs first, and then H2 O2 was added at once, or the mixture of MA and H2 O2 was added to Cit-AgNPs simultaneously, the color change was obvious even at 0.6 nM MA. It was worth noting that prolonging the interval time between adding MA and H2 O2 to Cit-AgNPs did not cause difference in the color change for CitAgNPs. However, if H2 O2 was added to Cit-AgNPs ahead of MA, the color change of the Cit-AgNPs was not as obvious as that when MA was added to Cit-AgNPs ahead of H2 O2 , or the mixture of MA and H2 O2 was added to Cit-AgNPs simultaneously. And the color change became more and more unobvious with the extension of
(1) (2)
MA molecule contained three exocyclic amino groups and three nitrogens in hybrid ring, which could be used as nucleophilic reagent (NR). A NR could donate its electron pair of N to the unoccupied orbital of AgNPs, leading to a small negative charge (␦− ) in the interior of the colloidal particles. As is known, the Fermi level of a metal is determined by the surface electron density of particles [29]. Due to the increase in the electron density caused by the adsorption of MA, the Fermi level of Cit-AgNPs shifted to a more negative potential (compared to Fermi potential at equilibrium). As a result, Cit-AgNPs became more reactive toward oxidation by electron acceptors due to their more negative potential and excess electrons. Excess electrons of AgNPs could be easily picked up by electron acceptors, forming Ag+ and reductive products of the electron acceptors [30,31]. In our experiment, H2 O2 was found to be a typical electron acceptor. Because MA also showed strong complexation ability to Ag+ [32], Ag+ generated was quickly complexed by MA to form the positive complex (AgM). Thus, elementary Ag was quickly etched away through forming AgM by an “oxidativeetching” process. Our study was in accordance with the previous studies that the oxidative etching of metal NPs could be accelerated in the presence of nucleophilic reagents and electron acceptors [23]. In the experiment, the cooperation effect of a nucleophilic reagent (MA), electron acceptor (H2 O2 ) and the strong complexation ability of the nucleophilic reagent with Ag+ , ultimately led to an effective “oxidative etching” process. Moreover, chemisorption of a positively charged complex AgM replaced the Cit molecules on the surface and reduced the overall surface negative charges of Cit-AgNPs, resulted in aggregation of AgNPs. The weakening, broadening and red-shift in the absorbance of Cit-AgNPs were characteristics of the formation of aggregation of metal NPs [33,34]. To confirm our presumption that the formed AgM complex played a key role in the detection of MA, different concentrations of AgM complex [32] made by mixing the equal molar ratio of Ag+ and MA were added to Cit-AgNPs to investigate the influence of AgM complex on the color and absorption spectra of Cit-AgNPs. As shown in Fig. S2, with the increase of the concentration of AgM, the extent of change in color and absorbance was also as obvious as that when the same concentration of MA and 0.6 mM H2 O2 were sequentially added to Cit-AgNPs (as shown in Fig. 1). However, the addition of MA (Fig. S1) or Ag+ (Fig. S3) alone could not cause the similar obvious absorption or color change of Cit-AgNPs. The above results confirmed that the formation of the complex of AgM was important for the aggregation of AgNPs and highly sensitive MA sensing.
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Fig. 2. UV–vis absorption spectra and photograph (A) and the corresponding particle size (black) and zeta potential (white) (B) of Cit-AgNPs (a), Cit-AgNPs after adding H2 O2 (0.6 mM) (b), Cit-AgNPs after adding MA (10 M) (c) and Cit-AgNPs after the addition of MA (10 M) and H2 O2 (0.6 mM) (d).
Fig. 3. The resonance light scattering spectra of the Cit-AgNPs at different concentrations of MA (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM) in the presence of 0.6 mM H2 O2 .
To further explore the mechanism of the method for the detection of MA, the products of Cit-AgNPs after reaction with different amounts of MA and 0.6 mM H2 O2 were characterized by dynamic light scattering (Table S2) and resonance light scattering spectra (Fig. 3). The decrease of the zeta potential from −28.75 mV to −1.32 mV indicated that more and more negative charges were lost on the surface of AgNPs after the reaction of AgNPs with the increased concentration of MA in the presence of H2 O2 . While the increase of the particle size from 76.5 nm to 382.6 nm as well as the enhanced resonance light scattering intensity demonstrated that more and more aggregates of AgNPs formed due to the loss of Cit on the surface induced by the reaction between AgNPs, MA and H2 O2 . The MA stimulated aggregation of AgNPs in the
presence of H2 O2 was also evidenced by HRTEM images, which revealed that monodisperse AgNPs became significantly aggregated in the presence of MA and H2 O2 (Fig. 4(b) and (c)). Based on the above analysis, an “oxidative etching-aggregation” mechanism was proposed for MA detection: the cooperation effect of the nucleophilic reagent (MA) and the electron acceptor (H2 O2 ) enhanced the reactivity (oxidation) of AgNPs and the strong complexation ability of the nucleophilic reagent and Ag+ promoted the etching of the AgNPs; the chemisorption of a positive complex (AgM) brought about aggregation of AgNPs. The “oxidative etchingaggregation” mechanism proposed for MA detection is depicted in Scheme 1. To certify the significant role of the electron acceptors in this study, H2 O2 was substituted by other electron acceptors such as ozone (Fig. S4) or Fe(NO3 )3 (Fig. S5) for the colorimetric detection of MA. After the Cit-AgNPs solution was bubbled for 15 s by ozone, color of the solution did not change and the absorption band only red-shifted slightly (data not shown), which was similar to that of the color/absorption change of Cit-AgNPs induced by H2 O2 . This was because that the reaction of ozone with AgNPs followed a similar reformation process as that of the reaction between H2 O2 and AgNPs [27,28]. With the increased concentration of MA, the variation of color and absorption of Cit-AgNPs in the presence of ozone was similar to that when H2 O2 was used as the electron acceptor (Fig. S4). Fe(III) species, a frequently-used etching reagent for noble metal NPs [35], was also used as an electron acceptor in this study (Fig. S5). As expected, the sensitivity of detecting MA by Cit-AgNPs could also be promoted by Fe(NO3 )3 , though the sensitivity for MA detection using Fe(NO3 )3 was not as high as that when using H2 O2 or ozone. This may be due to the lower oxidative potential of Fe(III) (E0 = 0.77 V) than that of H2 O2 (E0 = 1.77 V) and ozone (E0 = 2.07 V). From the above results, we can see that H2 O2 , ozone and Fe(NO3 )3 all could be used as effective electron acceptors to detect MA and
Fig. 4. The HRTEM images of Cit-AgNPs (a) and Cit-AgNPs after the addition of 0.6 mM H2 O2 in the absence (b) and presence (c) of 10 M of MA.
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Scheme 1. Schematic illustration of Cit-AgNPs probe for the assay of MA.
this method was versatile for MA detection using different electron acceptors. In addition, in the case of ozone or Fe(NO3 )3 , if melamine was added to Cit-AgNPs ahead of ozone/Fe(NO3 )3 , the detection of melamine was most sensitive. Exchanging the addition sequence of melamine and ozone/Fe(NO3 )3 or adding melamine and ozone/Fe(NO3 )3 to Cit-AgNPs at the same time, the sensitivity for MA detection would be reduced. These phenomena were consistent that in the case of H2 O2 , which could further confirm our detection mechanism. 3.2. MA and H2 O2 sensor design To investigate the effect of the reaction time on the “oxidative etching-aggregation” of AgNPs, the absorbance of Cit-AgNPs after adding MA and H2 O2 was recorded at different time intervals (Fig. S6). As can be seen, the reaction was completed after 25 min. Moreover, the effect of the reaction time on the “oxidative etchingaggregation” of AgNPs when using ozone or Fe(NO3 )3 was identical to that when using H2 O2 as electron acceptor. Therefore, 25 min was selected as the ideal reaction time for MA detection. It was evident that the Cit-AgNPs-MA-electron acceptor system could be used as ultrasensitive method for MA detection. As observed, upon adding H2 O2 or ozone to Cit-AgNPs with increased concentrations of MA, the color of Cit-AgNPs changed progressively from yellow, orange to blackish green (Fig. 1 and Fig. S4). While for Fe(NO3 )3 , the color of Cit-AgNPs changed from yellow to orange with the increased concentration of MA (Fig. S5). Even the addition of 0.6 nM (when using H2 O2 or ozone as electron acceptor) or 0.01 M (when using Fe(NO3 )3 as electron acceptor) of MA caused
a color change that could be unambiguously distinguished from that of the initial suspension of Cit-AgNPs. UV–vis spectra demonstrated that the A0 − A (A0 and A stand for the absorption intensity of AgNPs before and after reaction with MA in the presence of electron acceptors) value was linear with the logarithmic concentrations of MA in the range of 0.2 nM to 3 M, 0.3 nM to 3 M, and 5 nM to 10 M when using H2 O2 , ozone and Fe(NO3 )3 as electron acceptors, respectively (Fig. S7). The detection limits were as low as 0.08 nM, 0.16 nM, and 3.0 nM when H2 O2 , ozone or Fe(NO3 )3 was used as an electron acceptor, respectively. To the best of our knowledge, the detection limit obtained by using H2 O2 as electron acceptor was the lowest one in the current reported colorimetric methods to assay MA (Table S1). Selectivity is also a big challenge for the detection of MA [12]. As shown in Fig. 5, all the investigated interferences (vitaminC, tyrosine, alanine, tryptophan, methionine, glucose, glycerol, urea, CaCl2 , FeCl3 , KCl, MgCl2 , ZnSO4 , KBr, MnSO4 ) did not induce an obviously visual color change or absorbance variation of Cit-AgNPs. Using ozone or Fe(NO3 )3 as an electron acceptor also exhibited similar good specificity for MA detection as that when using H2 O2 as an electron acceptor. These results indicated that this system was appropriate for the selective recognition of MA. It was also found that the extent of “oxidative etchingaggregation”, namely the variation color and absorption of Cit-AgNPs could be controlled by varying the concentration of MA or the electron acceptors. So, the detection system could be potentially used as versatile functional sensors for MA and electron acceptors (H2 O2 , ozone or Fe(NO3 )3 ). Considering the pharmaceutical, clinical, environmental and biological importance of H2 O2
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concentrations of MA. In the presence of 6 M MA, the solution of Cit-AgNPs changed from yellow, orange to dark brown with the increased concentration of H2 O2 (Fig. 6). It was worth noting that the probe enabled the analysis of H2 O2 with a minimum detectable concentration that corresponded to 1 nM by naked-eye. To the best of our knowledge, this concentration was much lower than the reported colorimetric assays based on AuNPs [39,40] and the commonly used electrochemical methods to detect H2 O2 [41–43]. The A0 − A value increased linearly with the logarithmic concentrations of H2 O2 over the range from 0.6 nM to 5 M (R = 0.995), with a detection limit of 0.2 nM (Fig. 6). Moreover, the presence of 20 times of common interferences including glucose, uric acid, or ascorbic acid did not induce distinct absorption or color change for the determination of 0.2 M H2 O2 , indicating an acceptable selectivity of this assay for H2 O2 detection.
3.3. Colorimetric detection of MA and H2 O2 in real samples
Fig. 5. Selectivity of this assay. (top part) Color changes of the Cit-AgNPs solution by adding various representative interferences at concentrations of 2 M. (bottom part) The absorption changes (A0 − A) of Cit-AgNPs in the presence of different representative interferences at concentrations of 2 M.
[36–38], this AgNPs-MA-electron acceptor system was also used to detect H2 O2 at a fixed MA concentration. In our experiment, 6 M MA was chosen for H2 O2 detection because at this concentration MA alone did not induce change in color and absorbance of Cit-AgNPs (Fig. S1). Meanwhile, the sensitivity of the detection system for H2 O2 was much higher than that when using lower
The feasibility of this approach for sensing MA in milk sample and H2 O2 in rain water sample was evaluated. Upon the addition of MA at a concentration of 0.6 nM, which was much lower than the limit of 7.9 M in infant formula officially stipulated by the UN food standard commission [26], the color of the Cit-AgNPs solution turned from yellow to dark yellow (Fig. S8A) when H2 O2 was used as an electron acceptor. And 1 nM H2 O2 in rain water could also be distinguished by naked-eye using this method (Fig. S8B). Good linear correlations (R = 0.993 and 0.988) existed between the A0 − A and the logarithmic concentration of MA (0.3 nM to 3 M) (Fig. S9) or H2 O2 (0.7 nM to 3 M) (Fig. S10). Moreover, the recoveries were 104%, 103% and 97% for 50 nM, 100 nM and 500 nM of MA, respectively (Table 1). Compared the obtained recoveries in our work with that obtained by HPLC [44], the accuracy of our method was credible and this method could be applied successfully to the determination of MA in pretreated liquid milk products. To conclude, the method developed here was sufficient for rapid screening of MA in milk sample and H2 O2 in rain water with a simple, sensitive and selective manner.
Fig. 6. UV–vis spectra (bottom image, left) and visual color change (top image) of Cit-AgNPs traced from increasing amounts of H2 O2 in the presence of 6 M MA; a plot of the A0 − A versus the logarithmic concentrations of H2 O2 ranged from 0.6 nM to 5 M (bottom image, right).
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Table 1 Analytical results for melamine in milk samples. Milk samples
Added (nM)
Founda
Recovery (%)
RSD (%)
1 2 3
50 100 500
51.8 103.4 483.1
104 103 97
2.4 4.8 4.9
RSD: relative standard deviation. a The mean of three experiments.
4. Conclusions In summary, we designed a versatile colorimetric method using Cit-AgNPs to detect MA and H2 O2 that offered advantages of simplicity, rapidity, high sensitivity, and good selectivity. It was manifested that the presence of MA (acting as nucleophilic reagent for AgNPs and complex ligand for Ag+ ) and an electron acceptor such as H2 O2 , ozone or Fe(NO3 )3 could lead to fast and effective “oxidative etching-aggregation” of AgNPs which could be detected by naked-eye. The experimental results reported here also provided possibilities to detect other electron acceptors and other nucleophilic agents, thus paving the way for the wide range of new potential applications of AgNPs in the detection of other analytes in the future. Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 21005031 and 20903048), the Fundamental Research Funds for the Central Universities (JUSRP21113), the Opening Foundation of the State Key Laboratory of MaterialsOriented Chemical Engineering (KL10-08). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2012.08.019. References [1] Y. Zhou, S.X. Wang, K. Zhang, X.Y. Jiang, Angew. Chem. Int. Ed. 47 (2008) 7454–7456. [2] H.X. Lin, Y. Zou, Y.S. Huang, J. Chen, W.Y. Zhang, Z.X. Zhuang, G. Jenkins, C.J. Yang, Chem. Commun. 47 (2011) 9312–9314. [3] M. Zhang, B.C. Ye, Anal. Chem. 83 (2011) 1504–1509. [4] Y. Jiang, H. Zhao, N.N. Zhu, Y.Q. Lin, P. Yu, L.Q. Mao, Angew. Chem. Int. Ed. 47 (2008) 8601–8604. [5] Y. Yuan, J. Zhang, H.C. Zhan, X.R. Yang, Biosens. Bioelectron. 26 (2011) 4245–4248. [6] Y.H. Lin, C.E. Chen, C.Y. Wang, F. Pu, J.S. Ren, X.G. Qu, Chem. Commun. 47 (2011) 1181–1183.
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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca
“Oxidative etching-aggregation” of silver nanoparticles by melamine and electron acceptors: An innovative route toward ultrasensitive and versatile functional colorimetric sensors Guang-Li Wang a,∗ , Xiao-Ying Zhu a , Huan-Jun Jiao a , Yu-Ming Dong a,b , Xiu-Ming Wu a , Zai-Jun Li a a b
The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing University of Technology, Nanjing 210009, PR China
h i g h l i g h t s
g r a p h i c a l
a b s t r a c t
A novel concept for colorimetric sensing of MA based on non-crosslinking of AgNPs. The detection mechanism was based on “oxidative etching-aggregation” of unmodified AgNPs. This method was simple, selective and ultrasensitive. The detection limit for MA was as low as 0.08 nM when using H2 O2 as electron acceptor. The method also enabled the detection of H2 O2 with a detection limit of 0.2 nM.
a r t i c l e
i n f o
Article history: Received 5 June 2012 Received in revised form 11 August 2012 Accepted 16 August 2012 Available online 23 August 2012 Keywords: Colorimetric sensor Melamine Silver nanoparticles Oxidative etching-aggregation
a b s t r a c t An innovative and versatile functional colorimetric sensor for melamine (MA) and H2 O2 was developed with simplicity, excellent selectivity and ultrasensitivity. The detection mechanism was based on the “oxidative etching-aggregation” of silver nanoparticles (AgNPs) by the cooperation effect of MA and electron acceptors such as H2 O2 , ozone or Fe(NO3 )3 . The detection limits of this method for MA could reach as low as 0.08 nM, 0.16 nM and 3 nM when H2 O2 , ozone or Fe(NO3 )3 was used as an electron acceptor, respectively. When using H2 O2 as a typical electron acceptor, the method enabled the detection of H2 O2 with a detection limit of 0.2 nM. This proposed method offered a new way to design MA and H2 O2 sensors and might be easily extended to detect other nucleophilic reagents and electron acceptors based on colorimetric sensors. © 2012 Elsevier B.V. All rights reserved.
1. Introduction With the development of nanoscience and nanotechnology, more and more opportunities are provided for the application of nanomaterials in colorimetric sensing. Especially, gold nanoparticles (AuNPs) and silver nanoparticles (AgNPs) have emerged
∗ Corresponding author. Tel.: +86 510 85917090; fax: +86 510 85917763. E-mail address: [email protected] (G.-L. Wang). 0003-2670/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.aca.2012.08.019
as exciting materials as colorimetric probes because they have strong surface plasmon resonance and behavior that depends on the interparticle distance [1–4]. When the distances between the AuNPs/AgNPs become less than the average particle diameter, aggregation of the dispersed NPs occurred. Interparticle plasmon coupling of AuNPs/AgNPs occurred in aggregated NPs and the color of the NPs’ solution changed, which enabled the development of sensitive colorimetric sensors [5–7]. Recently, assays to detect melamine (MA) based on interparticle crosslinking aggregation by hydrogen-bonding or charge-transfer recognition between MA and
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surface modifiers of AuNPs were reported [8–18]. Although using surface modifiers to functionalize NPs is a successful tool to endow NPs with the recognition ability for MA, it makes the preparation of the NPs cumbersome and the detection procedure complicated. Moreover, surface modification usually deteriorates the superior optical properties of the metal NPs. Hence, it is desirable to develop an analytical method for the simple, sensitive and selective detection of MA without surface modification. It is worth noting that, for colorimetric assays, AgNPs have some advantages over AuNPs because they are cheaper and possess higher extinction coefficients compared to AuNPs with the same size [19]. In addition, AgNPs have reaction activeness though bulk Ag material is regarded as being inert. Herein, a novel concept for colorimetric detection of MA based on “oxidative etching-aggregation” of unmodified AgNPs with simplicity and ultrasensitivity was proposed. It was found that “oxidative etchingaggregation” of AgNPs could be very effective under the coexistence of MA with very low concentration (even at sub-nanomolar level) and electron acceptors. MA as a nucleophilic reagent could donate its electron pairs of N to the unoccupied orbital of AgNPs, which led to a more negative Fermi potential of AgNPs with excess electrons. Thus, AgNPs were more active toward oxidation by electron acceptors. In addition, MA could strongly complex with Ag+ produced by the oxidative etching of AgNPs through MA and electron acceptors. Chemisorption of the positively charged complex made of Ag+ and MA would lead to the decrease of the surface charges, resulting in non-crosslinking aggregation and significant change in color of the AgNPs. This method offered a new way to design MA sensors. More importantly, this work also provides possibilities to detect electron acceptors. Based on the above consideration, we demonstrated herein a simple and versatile functional colorimetric sensor for MA and H2 O2 with good selectivity and ultrasensitivity. This method opens up a new perspective for the development of highly sensitive colorimetric sensors based on the special reactivity of AgNPs with nucleophilic reagents and electron acceptors. 2. Experimental details 2.1. Chemical reagents Melamine, silver nitrate, trisodium citrate, hydrogen peroxide (30 wt%), sodium borohydride (96%), ferric nitrate were all obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All other chemicals used were of analytical grade. All solutions were prepared with ultrapure water (18.2 M cm−1 ) obtained from a Healforce water purification system. 2.2. Apparatus UV–vis absorption spectra were carried out using a TU-1901 spectrophotometer (Beijing Purkinje General Instrument Co., Ltd., Beijing, China). Photographs of the solutions were taken with Canon 530A digital camera. Resonance light scattering spectra were measured on a Cary Eclipse spectrofluorimeter (Varian, USA). The zeta potential and size distribution of the AgNPs were measured using a ZetaPALS zeta potential and particle size analyzer (Brookhaven, USA). High resolution transmission electron microscopy (HRTEM) images of AgNPs were obtained on a JEOL JEM-2100 transmission electron microscope (Hitachi, Japan). 2.3. Synthesis of Cit-AgNPs Citrate-capped AgNPs (Cit-AgNPs) were prepared by means of the chemical reduction of AgNO3 in the liquid phase according to the reported method [20] with modifications. To achieve this,
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20 mL of 1.0 mM AgNO3 was added to a 50 mL of solution containing 5.3 mM NaBH4 and 1.0 mM trisodium citrate. Then the mixture was allowed to react at room temperature for 18 h. 2.4. Colorimetric detection of MA and H2 O2 For MA sensing, electron acceptor (0.3 mL of 0.01 M H2 O2 or ozone (bubbled for 15 s) or 0.2 mL of 1 mM Fe(NO3 )3 ) was introduced to a solution containing 2 mL of Cit-AgNPs and certain amount of MA in a 5 mL calibrated test tube. Then the mixed solution was dissolved to volume with ultrapure water. The final concentrations were 0.6 mM, 0.42 mM and 0.04 mM for H2 O2 , ozone and Fe(NO3 )3 respectively. Twenty-five minutes later, the absorption spectra of the reaction mixture were recorded. Ozone was generated in a laboratory ozone generator using pure oxygen as gas source. The flow rate of oxygen was 15 mL min−1 , and the flow rate of ozone was 0.40 mg min−1 . To detect H2 O2 , different concentrations of H2 O2 were added to the solution containing 2 mL of Cit-AgNPs and 0.3 mL of 0.1 mM MA in a 5 mL calibrated test tube. Then the mixed solution was dissolved to volume with ultrapure water. The final concentration of MA was 6 M. 2.5. Colorimetric detection of MA and H2 O2 in real samples The method was used to detect MA in raw milk by standard addition method. The raw milk was pretreated as reported procedures [21]. The raw milk was purchased from local supermarkets. First, 1 mL of acetonitrile, 1 mL of CCl3 COOH, and 7 mL of water were added into 2.0 mL of raw milk. Then the mixture in a centrifuge tube was ultrasonically extracted for 20 min and then centrifugated at 5000 rpm for 20 min. The obtained supernatant was filtered two times. The obtained supernatant was adjusted to pH 8.0 with a small amount of 1 M NaOH. To quantify MA in raw milk, different amounts of MA were added to 2.0 mL of MA-free raw milk, then the mixture was handled as the steps described in the above sample preparation section. The obtained solutions were mixed with 2 mL of Cit-AgNPs before adding H2 O2 (0.3 mL, 0.01 M). To detect H2 O2 in rain water, a series of different concentrations of H2 O2 samples were prepared by “spiking” the rain water with different amounts of H2 O2 . The H2 O2 solution was added to a solution containing 2 mL of Cit-capped AgNPs and 0.3 mL of 0.1 mM MA. 3. Results and discussion 3.1. Colorimetric sensing mechanism The suspension of the as-synthesized Cit-AgNPs was yellow and transparent (Fig. S1), exhibited a characteristic and intense absorption peak around 400 nm owing to the surface plasmon excitation [22], which indicated a good dispersity of Cit-AgNPs in water. The negative charges of Cit on the surface of AgNPs acquired an electrostatic double layer, which provided a repulsive force between separated AgNPs and enabled the AgNPs to be dispersed stable in aqueous solution [23]. At first, Cit-AgNPs were directly utilized to quantify MA in aqueous solution. It was found that the color or the absorption of Cit-AgNPs did not change until the concentration of MA reached 10 M (Fig. S1). Through the coordinating interactions, nucleophilic primary amines as well as the ring nitrogens in hybrid aromatics of MA can bind onto the surface of metal NPs which are coordinatively unsaturated [24]. The adsorption of MA replaced the original weakly surface-bound citrate ions, neutralized the negative charge of Cit-AgNPs and induced aggregation of AgNPs. Particle aggregation led to color and absorption changes of
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the interval time between the addition of H2 O2 and MA into CitAgNPs. Considering that there was no reaction between MA and H2 O2 , a hypothesis was provided that intermediate species produced from the reaction of Cit-AgNPs and H2 O2 could interact with MA and played crucial role in the color change of Cit-AgNPs. Interestingly, it has been recently manifested that AgNPs catalyzed the decomposition of H2 O2 through a reformation process of AgNPs [27,28]. The reaction between AgNPs and H2 O2 generated superoxide anion and Ag+ (Eq. (1)). Ag+ could be quickly reduced to elementary Ag by superoxide anion and electron charged AgNPs (AgNP*− ) whose formation was mediated by the superoxide anion (Eq. (2)), resulting in the regeneration of AgNPs [27,28]. The redshift of the absorption band and slight absorption decrease of AgNPs after reaction with H2 O2 may be due to the particle size change of AgNPs resulted from the reformation process. Dynamic light scattering (DLS) result indicated that the average diameter of AgNPs increased from 74.8 nm to 76.5 nm after reaction with H2 O2 . •
AgNP + H2 O2 → Ag+ + O2− + 2H2 O •
AgNP + O2− → AgNP∗− + 2H2 O
Fig. 1. The photograph (top part) and evolution of absorption profiles (bottom part) of Cit-AgNPs in the presence of different concentrations of MA and 0.6 mM H2 O2 .
Cit-AgNPs [25]. Nevertheless, the color change induced by MA alone without any extra aid only occurred until the concentration of MA reached 10 M, which was higher than the safety limit (7.9 M for infant formula) defined by the UN food standard commission [26]. Exhilaratingly, upon the introduction of 0.6 mM H2 O2 into the mixture of Cit-AgNPs and MA, color change of Cit-AgNPs could be found even at 0.6 nM of MA (Fig. 1). To the best of our knowledge, this was the lowest concentration that could be detected with naked-eye using AuNPs or AgNPs (Table S1). However, it was found that the introduction of 0.6 mM H2 O2 alone could not induce any detectable color change of Cit-AgNPs (Fig. 2(b)). The absorption band of AgNPs red-shifted slightly from 397 nm to 400 nm and the absorption intensity weakened by a little (about 4.39%) after reaction with 0.6 mM H2 O2 (Fig. 2(b)). That is, the changes in color and absorption of Cit-AgNPs solution induced by the coexistence of H2 O2 and MA were much more obvious than the changes in color and absorption of Cit-AgNPs solution induced by H2 O2 or MA alone (Fig. 2b–d). Particle size and zeta potential measurements also confirmed the larger extent of changes in particle size and zeta potential of Cit-AgNPs in the coexistence of MA and H2 O2 than that in the presence of MA or H2 O2 alone (Fig. 2B), which was in accordance with the much more obvious color and absorption changes of Cit-AgNPs under the same experimental conditions. Hence, it should be pointed out that the color change in Fig. 1 should be ascribed to the cooperation effect of MA and H2 O2 . The adding ways of MA and H2 O2 to Cit-AgNPs solution also significantly affected the color change of Cit-AgNPs. When MA was added to Cit-AgNPs first, and then H2 O2 was added at once, or the mixture of MA and H2 O2 was added to Cit-AgNPs simultaneously, the color change was obvious even at 0.6 nM MA. It was worth noting that prolonging the interval time between adding MA and H2 O2 to Cit-AgNPs did not cause difference in the color change for CitAgNPs. However, if H2 O2 was added to Cit-AgNPs ahead of MA, the color change of the Cit-AgNPs was not as obvious as that when MA was added to Cit-AgNPs ahead of H2 O2 , or the mixture of MA and H2 O2 was added to Cit-AgNPs simultaneously. And the color change became more and more unobvious with the extension of
(1) (2)
MA molecule contained three exocyclic amino groups and three nitrogens in hybrid ring, which could be used as nucleophilic reagent (NR). A NR could donate its electron pair of N to the unoccupied orbital of AgNPs, leading to a small negative charge (␦− ) in the interior of the colloidal particles. As is known, the Fermi level of a metal is determined by the surface electron density of particles [29]. Due to the increase in the electron density caused by the adsorption of MA, the Fermi level of Cit-AgNPs shifted to a more negative potential (compared to Fermi potential at equilibrium). As a result, Cit-AgNPs became more reactive toward oxidation by electron acceptors due to their more negative potential and excess electrons. Excess electrons of AgNPs could be easily picked up by electron acceptors, forming Ag+ and reductive products of the electron acceptors [30,31]. In our experiment, H2 O2 was found to be a typical electron acceptor. Because MA also showed strong complexation ability to Ag+ [32], Ag+ generated was quickly complexed by MA to form the positive complex (AgM). Thus, elementary Ag was quickly etched away through forming AgM by an “oxidativeetching” process. Our study was in accordance with the previous studies that the oxidative etching of metal NPs could be accelerated in the presence of nucleophilic reagents and electron acceptors [23]. In the experiment, the cooperation effect of a nucleophilic reagent (MA), electron acceptor (H2 O2 ) and the strong complexation ability of the nucleophilic reagent with Ag+ , ultimately led to an effective “oxidative etching” process. Moreover, chemisorption of a positively charged complex AgM replaced the Cit molecules on the surface and reduced the overall surface negative charges of Cit-AgNPs, resulted in aggregation of AgNPs. The weakening, broadening and red-shift in the absorbance of Cit-AgNPs were characteristics of the formation of aggregation of metal NPs [33,34]. To confirm our presumption that the formed AgM complex played a key role in the detection of MA, different concentrations of AgM complex [32] made by mixing the equal molar ratio of Ag+ and MA were added to Cit-AgNPs to investigate the influence of AgM complex on the color and absorption spectra of Cit-AgNPs. As shown in Fig. S2, with the increase of the concentration of AgM, the extent of change in color and absorbance was also as obvious as that when the same concentration of MA and 0.6 mM H2 O2 were sequentially added to Cit-AgNPs (as shown in Fig. 1). However, the addition of MA (Fig. S1) or Ag+ (Fig. S3) alone could not cause the similar obvious absorption or color change of Cit-AgNPs. The above results confirmed that the formation of the complex of AgM was important for the aggregation of AgNPs and highly sensitive MA sensing.
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Fig. 2. UV–vis absorption spectra and photograph (A) and the corresponding particle size (black) and zeta potential (white) (B) of Cit-AgNPs (a), Cit-AgNPs after adding H2 O2 (0.6 mM) (b), Cit-AgNPs after adding MA (10 M) (c) and Cit-AgNPs after the addition of MA (10 M) and H2 O2 (0.6 mM) (d).
Fig. 3. The resonance light scattering spectra of the Cit-AgNPs at different concentrations of MA (0 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM) in the presence of 0.6 mM H2 O2 .
To further explore the mechanism of the method for the detection of MA, the products of Cit-AgNPs after reaction with different amounts of MA and 0.6 mM H2 O2 were characterized by dynamic light scattering (Table S2) and resonance light scattering spectra (Fig. 3). The decrease of the zeta potential from −28.75 mV to −1.32 mV indicated that more and more negative charges were lost on the surface of AgNPs after the reaction of AgNPs with the increased concentration of MA in the presence of H2 O2 . While the increase of the particle size from 76.5 nm to 382.6 nm as well as the enhanced resonance light scattering intensity demonstrated that more and more aggregates of AgNPs formed due to the loss of Cit on the surface induced by the reaction between AgNPs, MA and H2 O2 . The MA stimulated aggregation of AgNPs in the
presence of H2 O2 was also evidenced by HRTEM images, which revealed that monodisperse AgNPs became significantly aggregated in the presence of MA and H2 O2 (Fig. 4(b) and (c)). Based on the above analysis, an “oxidative etching-aggregation” mechanism was proposed for MA detection: the cooperation effect of the nucleophilic reagent (MA) and the electron acceptor (H2 O2 ) enhanced the reactivity (oxidation) of AgNPs and the strong complexation ability of the nucleophilic reagent and Ag+ promoted the etching of the AgNPs; the chemisorption of a positive complex (AgM) brought about aggregation of AgNPs. The “oxidative etchingaggregation” mechanism proposed for MA detection is depicted in Scheme 1. To certify the significant role of the electron acceptors in this study, H2 O2 was substituted by other electron acceptors such as ozone (Fig. S4) or Fe(NO3 )3 (Fig. S5) for the colorimetric detection of MA. After the Cit-AgNPs solution was bubbled for 15 s by ozone, color of the solution did not change and the absorption band only red-shifted slightly (data not shown), which was similar to that of the color/absorption change of Cit-AgNPs induced by H2 O2 . This was because that the reaction of ozone with AgNPs followed a similar reformation process as that of the reaction between H2 O2 and AgNPs [27,28]. With the increased concentration of MA, the variation of color and absorption of Cit-AgNPs in the presence of ozone was similar to that when H2 O2 was used as the electron acceptor (Fig. S4). Fe(III) species, a frequently-used etching reagent for noble metal NPs [35], was also used as an electron acceptor in this study (Fig. S5). As expected, the sensitivity of detecting MA by Cit-AgNPs could also be promoted by Fe(NO3 )3 , though the sensitivity for MA detection using Fe(NO3 )3 was not as high as that when using H2 O2 or ozone. This may be due to the lower oxidative potential of Fe(III) (E0 = 0.77 V) than that of H2 O2 (E0 = 1.77 V) and ozone (E0 = 2.07 V). From the above results, we can see that H2 O2 , ozone and Fe(NO3 )3 all could be used as effective electron acceptors to detect MA and
Fig. 4. The HRTEM images of Cit-AgNPs (a) and Cit-AgNPs after the addition of 0.6 mM H2 O2 in the absence (b) and presence (c) of 10 M of MA.
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Scheme 1. Schematic illustration of Cit-AgNPs probe for the assay of MA.
this method was versatile for MA detection using different electron acceptors. In addition, in the case of ozone or Fe(NO3 )3 , if melamine was added to Cit-AgNPs ahead of ozone/Fe(NO3 )3 , the detection of melamine was most sensitive. Exchanging the addition sequence of melamine and ozone/Fe(NO3 )3 or adding melamine and ozone/Fe(NO3 )3 to Cit-AgNPs at the same time, the sensitivity for MA detection would be reduced. These phenomena were consistent that in the case of H2 O2 , which could further confirm our detection mechanism. 3.2. MA and H2 O2 sensor design To investigate the effect of the reaction time on the “oxidative etching-aggregation” of AgNPs, the absorbance of Cit-AgNPs after adding MA and H2 O2 was recorded at different time intervals (Fig. S6). As can be seen, the reaction was completed after 25 min. Moreover, the effect of the reaction time on the “oxidative etchingaggregation” of AgNPs when using ozone or Fe(NO3 )3 was identical to that when using H2 O2 as electron acceptor. Therefore, 25 min was selected as the ideal reaction time for MA detection. It was evident that the Cit-AgNPs-MA-electron acceptor system could be used as ultrasensitive method for MA detection. As observed, upon adding H2 O2 or ozone to Cit-AgNPs with increased concentrations of MA, the color of Cit-AgNPs changed progressively from yellow, orange to blackish green (Fig. 1 and Fig. S4). While for Fe(NO3 )3 , the color of Cit-AgNPs changed from yellow to orange with the increased concentration of MA (Fig. S5). Even the addition of 0.6 nM (when using H2 O2 or ozone as electron acceptor) or 0.01 M (when using Fe(NO3 )3 as electron acceptor) of MA caused
a color change that could be unambiguously distinguished from that of the initial suspension of Cit-AgNPs. UV–vis spectra demonstrated that the A0 − A (A0 and A stand for the absorption intensity of AgNPs before and after reaction with MA in the presence of electron acceptors) value was linear with the logarithmic concentrations of MA in the range of 0.2 nM to 3 M, 0.3 nM to 3 M, and 5 nM to 10 M when using H2 O2 , ozone and Fe(NO3 )3 as electron acceptors, respectively (Fig. S7). The detection limits were as low as 0.08 nM, 0.16 nM, and 3.0 nM when H2 O2 , ozone or Fe(NO3 )3 was used as an electron acceptor, respectively. To the best of our knowledge, the detection limit obtained by using H2 O2 as electron acceptor was the lowest one in the current reported colorimetric methods to assay MA (Table S1). Selectivity is also a big challenge for the detection of MA [12]. As shown in Fig. 5, all the investigated interferences (vitaminC, tyrosine, alanine, tryptophan, methionine, glucose, glycerol, urea, CaCl2 , FeCl3 , KCl, MgCl2 , ZnSO4 , KBr, MnSO4 ) did not induce an obviously visual color change or absorbance variation of Cit-AgNPs. Using ozone or Fe(NO3 )3 as an electron acceptor also exhibited similar good specificity for MA detection as that when using H2 O2 as an electron acceptor. These results indicated that this system was appropriate for the selective recognition of MA. It was also found that the extent of “oxidative etchingaggregation”, namely the variation color and absorption of Cit-AgNPs could be controlled by varying the concentration of MA or the electron acceptors. So, the detection system could be potentially used as versatile functional sensors for MA and electron acceptors (H2 O2 , ozone or Fe(NO3 )3 ). Considering the pharmaceutical, clinical, environmental and biological importance of H2 O2
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concentrations of MA. In the presence of 6 M MA, the solution of Cit-AgNPs changed from yellow, orange to dark brown with the increased concentration of H2 O2 (Fig. 6). It was worth noting that the probe enabled the analysis of H2 O2 with a minimum detectable concentration that corresponded to 1 nM by naked-eye. To the best of our knowledge, this concentration was much lower than the reported colorimetric assays based on AuNPs [39,40] and the commonly used electrochemical methods to detect H2 O2 [41–43]. The A0 − A value increased linearly with the logarithmic concentrations of H2 O2 over the range from 0.6 nM to 5 M (R = 0.995), with a detection limit of 0.2 nM (Fig. 6). Moreover, the presence of 20 times of common interferences including glucose, uric acid, or ascorbic acid did not induce distinct absorption or color change for the determination of 0.2 M H2 O2 , indicating an acceptable selectivity of this assay for H2 O2 detection.
3.3. Colorimetric detection of MA and H2 O2 in real samples
Fig. 5. Selectivity of this assay. (top part) Color changes of the Cit-AgNPs solution by adding various representative interferences at concentrations of 2 M. (bottom part) The absorption changes (A0 − A) of Cit-AgNPs in the presence of different representative interferences at concentrations of 2 M.
[36–38], this AgNPs-MA-electron acceptor system was also used to detect H2 O2 at a fixed MA concentration. In our experiment, 6 M MA was chosen for H2 O2 detection because at this concentration MA alone did not induce change in color and absorbance of Cit-AgNPs (Fig. S1). Meanwhile, the sensitivity of the detection system for H2 O2 was much higher than that when using lower
The feasibility of this approach for sensing MA in milk sample and H2 O2 in rain water sample was evaluated. Upon the addition of MA at a concentration of 0.6 nM, which was much lower than the limit of 7.9 M in infant formula officially stipulated by the UN food standard commission [26], the color of the Cit-AgNPs solution turned from yellow to dark yellow (Fig. S8A) when H2 O2 was used as an electron acceptor. And 1 nM H2 O2 in rain water could also be distinguished by naked-eye using this method (Fig. S8B). Good linear correlations (R = 0.993 and 0.988) existed between the A0 − A and the logarithmic concentration of MA (0.3 nM to 3 M) (Fig. S9) or H2 O2 (0.7 nM to 3 M) (Fig. S10). Moreover, the recoveries were 104%, 103% and 97% for 50 nM, 100 nM and 500 nM of MA, respectively (Table 1). Compared the obtained recoveries in our work with that obtained by HPLC [44], the accuracy of our method was credible and this method could be applied successfully to the determination of MA in pretreated liquid milk products. To conclude, the method developed here was sufficient for rapid screening of MA in milk sample and H2 O2 in rain water with a simple, sensitive and selective manner.
Fig. 6. UV–vis spectra (bottom image, left) and visual color change (top image) of Cit-AgNPs traced from increasing amounts of H2 O2 in the presence of 6 M MA; a plot of the A0 − A versus the logarithmic concentrations of H2 O2 ranged from 0.6 nM to 5 M (bottom image, right).
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Table 1 Analytical results for melamine in milk samples. Milk samples
Added (nM)
Founda
Recovery (%)
RSD (%)
1 2 3
50 100 500
51.8 103.4 483.1
104 103 97
2.4 4.8 4.9
RSD: relative standard deviation. a The mean of three experiments.
4. Conclusions In summary, we designed a versatile colorimetric method using Cit-AgNPs to detect MA and H2 O2 that offered advantages of simplicity, rapidity, high sensitivity, and good selectivity. It was manifested that the presence of MA (acting as nucleophilic reagent for AgNPs and complex ligand for Ag+ ) and an electron acceptor such as H2 O2 , ozone or Fe(NO3 )3 could lead to fast and effective “oxidative etching-aggregation” of AgNPs which could be detected by naked-eye. The experimental results reported here also provided possibilities to detect other electron acceptors and other nucleophilic agents, thus paving the way for the wide range of new potential applications of AgNPs in the detection of other analytes in the future. Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 21005031 and 20903048), the Fundamental Research Funds for the Central Universities (JUSRP21113), the Opening Foundation of the State Key Laboratory of MaterialsOriented Chemical Engineering (KL10-08). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2012.08.019. References [1] Y. Zhou, S.X. Wang, K. Zhang, X.Y. Jiang, Angew. Chem. Int. Ed. 47 (2008) 7454–7456. [2] H.X. Lin, Y. Zou, Y.S. Huang, J. Chen, W.Y. Zhang, Z.X. Zhuang, G. Jenkins, C.J. Yang, Chem. Commun. 47 (2011) 9312–9314. [3] M. Zhang, B.C. Ye, Anal. Chem. 83 (2011) 1504–1509. [4] Y. Jiang, H. Zhao, N.N. Zhu, Y.Q. Lin, P. Yu, L.Q. Mao, Angew. Chem. Int. Ed. 47 (2008) 8601–8604. [5] Y. Yuan, J. Zhang, H.C. Zhan, X.R. Yang, Biosens. Bioelectron. 26 (2011) 4245–4248. [6] Y.H. Lin, C.E. Chen, C.Y. Wang, F. Pu, J.S. Ren, X.G. Qu, Chem. Commun. 47 (2011) 1181–1183.
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