Melamine sensing through riboflavin stabilized gold nanoparticles

Abstract

Melamine (M) sensing has been achieved through supramolecular assembly with riboflavin (R) via H-bonding in the platform of R stabilized gold nanoparticles (R-Au NPs), by colorimetric as well as UV-vis techniques.

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Melamine, widely used in the manufacturing of polymer resins, is unethically used by milk manufacturers in adulterating milk to make it appear more protein-rich, due the high nitrogen content of melamine. This creates severe renal disease for both humans and animals due to the formation of insoluble melamine cyanurate crystals in the kidneys.^1–5 A certain maximum level of melamine ingestion is fixed in infant foods, e.g. 2.5 ppm in the USA and 1 ppm in China.⁶ So, there is an urgent need for the development of a simple, reliable and easily accessible tool to detect and quantitatively measure the melamine contamination in milk food with a high sensitivity. Gas chromatography/mass spectrometry (GC-MS),^7agas chromatography,^7bliquid chromatography/mass spectrometry (LC-MS),^7ccapillary electrophoresis,^7dmatrix-assisted laser desorption ionization (MALDI) MS^7e and enzyme-linked immunosorbent assay (ELISA)^7f have been developed for the detection of melamine. Recently, the use of the surface plasmon properties of Au and Ag nanoparticles in the pristine and aggregated state for the colorimetric detection of melamine was reported.^6,8,9 We have reported the self-assembly of riboflavin (vitamin B2; R) with melamine (M) to produce a hydrogel¹⁰via a supramolecular complex (Scheme 1) formation through H-bonding. Now, we are also able to produce R stabilized gold nanoparticles (R-AuNPs) in aqueous medium and this may help to develop an easier method to detect melamine in foods using the surface plasmon property of Au.

Scheme 1 Schematic presentation of the stabilization of Au nanoparticles (green sphere) by complexation of adsorbed Au³⁺ with riboflavin (R) followed by destabilization with melamine (M) which produces the RM31 supramolecular complex, destabilizing the Au nanoparticles.

In this communication we report melamine recognition on the basis of supramolecular assembly of riboflavin (vitamin B-2) with melaminevia H-bonding in the platform of R-stabilized gold nanoparticles (R-Au NPs) by a simple colorimetric technique. Au NPs are prepared by adding drop wise a freshly prepared 1 mM sodium borohydride (NaBH₄) solution into 1 mM HAuCl₄, 3H₂O solutions (1 : 1 ratio by vol) with a constant stirring for 1 h. The yellow color of gold chloride changes into a pink color due to the addition of borohydride. Ethylene glycol (EG, 5–10 equivalents) is initially added to the gold chloride solution to act as a stabilizer. The freshly prepared Au-NP solution (5 × 10⁻⁵ M) is mixed with a 1 mM solution of R in a culture tube and diluted with water to make the concentration of both Au NP and R 1 μM. The mixed solution is then stirred for 5 h in a dark place at 25 °C with a magnetic stirrer. The color of the R-Au NP solution remains pink and it is stable for more than a week when stored in a dark place. The formation of Au NPs is confirmed from the UV-vis spectra showing the plasmon band at 535 nm. There is no shift of the UV-vis peak of R-Au NPs from that of the glycol stabilized Au NP solution (Fig. 1).

Fig. 1 UV-vis spectra of Au NPs, R-Au NPs and melamine sensed R-Au NPs. Inset: visual color change of R-Au NPs upon addition of melamine (from left to right: 0, 0.1, 0.25, 0.5 and 1 μM).

In the inset of Fig. 1 the color change of the solutions from pure R-Au NP (bottle 1) to different M concentrations of 0.1 to 1 μM (bottles 2–5) present in the bottles is shown. The color change is significant from bottle 3 to bottle 4, i.e. at the concentration of 0.25–0.5 μM melamine. So, the naked eye observation makes it a useful colorimetric detection method at a concentration of M below the safety level in foods. The color change is also monitored from UV-vis spectra (Fig. 1 and supplementary Fig. S1†) where the 0.5 μM melamine containing solution exhibits a new peak at 732 nm. The intensity of the plasmon peak at 535 nm has diminished from the initial value. The spectra in Fig. S1 indicate that there is no absorption peak of R or RM complex at the 535 nm and 732 nm regions facilitating the melamine sensor activity of R-Au NPs. The cause of the formation of a new peak at 732 nm may be obtained from the TEM micrographs presented in Fig. 2 where the sizes of Au nanoparticles are compared. Both the EG and R stabilized nanoparticles have a similar shape and size (Fig. 2a and 2b) but on addition of M, agglomerated Au nanoparticles are observed (Fig. 2c). These agglomerated Au nanoparticles produce a longitudinal plasmon band at 732 nm and the 535 nm band shows a red shift to 540 nm due to an increase in lateral size. Thus it may be argued that the two plasmon bands at 540 nm and 732 nm in the solution containing 0.5 μM of M is due to the transverse and longitudinal mode of the surface plasmon band, respectively.¹¹ The mechanism of coalescence of Au nanoparticles will be discussed here.

Fig. 2 TEM images of (a) glycol stabilized Au NPs (b) R-Au NPs, (c) melanine (1.5 μM) recognized R-Au NPs and (d) supramolecular network structure of RM complex containing Au NPs.

It is a usual phenomenon that nanoparticles adsorb the parent ions on its surface,¹² so Au³⁺ ions would remain adsorbed on Au nanoparticles. In the EG stabilized system the adsorbed Au³⁺ ions produce complexes with hydroxyl groups of EG and thus prevent coalescence from other Au nanoparticles. But on addition of R the EG is replaced by the stronger complexing agent R (through the >C=O and –NH group), thus stabilizing the Au nanoparticles.¹³ On addition of M it forms supramolecular complexes with R (Scheme 1) making free Au NPs which coalesce due to their strong cohesive force. From the intensity vs. time plots of both the 535 and 732 nm peaks (Fig. S2, ESI†) it is evident that the 732 nm peak is not produced at the expense of the 535 nm peak as the intensity of the latter does not decrease much compared to the rise of intensity of the 732 nm peak. Hence it may be concluded that coalescence of nanoparticles is the cause of the formation of the new 732 nm peak. The coalescence of Au nanoparticles is manifested from the black color of the solution and the formation of an additional 732 nm peak in the UV-vis spectra. The process is almost instantaneous (2 mins waiting time) indicating dynamic reversibility of the supramolecular interactions.¹⁴ The complexation of R and M is evidenced from the TEM micrograph (Fig. 2d) where a fibrillar network is observed.¹⁰ To support the above assertion, FTIR spectra of the components and R stabilized Au nanoparticles (R-Au NPs) and their change on addition of M are presented in Fig. 3. It is apparent from the figure that the 1733 cm⁻¹ peak of the >C=O group of R is not observed at all in the R stabilized Au nanoparticles, indicating the >C=O group produces a complex with Au³⁺ ions adsorbed on Au nanoparticles through the oxygen atom. The peaks at 3200–3500 cm⁻¹ are due to N–H vibration peaks¹⁰ and these are lost in the spectrum of R-Au NPs, indicating that the imino nitrogens of R are also involved in the complex formation, stabilizing the Au nanoparticles (Scheme 1).¹³ On addition of M the 1733 cm⁻¹ peak of R shows a shift to 1683 cm⁻¹ indicating H-bonding interaction with the amino group of M. The 3419 and 3469 cm⁻¹ peaks of M are not at all prominent indicating H-bonding interaction of M with R. Hence, the FTIR results support the assertion that on addition of M to R-Au NPs, RM complexation occurs making the Au nanoparticles less stabilized, causing their agglomeration.

Fig. 3 Comparison of FTIR spectra of R-Au NPs and R-M Au NPs with those of pure R and M.

In the development of an optimum sensing system we first examine the minimum concentration of capping agent that could be used to detect a certain melamine concentration. So we use several stocks of 2 ml glycol stabilized Au NP solution (final concentration = 1 μM) containing different volumes from 20 μl to 200 μl R (1 × 10⁻⁴ M solutions) and the solutions are kept for 5 h in the dark at 25 °C to complete the capping process of R by exchanging EG. A constant amount (20 μl) of melamine solution (final concentration = 1 μM) is added in each stock and UV-vis spectra are recorded after 10 min. The sensitivity is determined by the absorbance ratio of the newly generated peak (732 nm) intensity with that of the 535 nm peak intensity. It is apparent from Fig. S3 (ESI†) that a leveling of sensitivity (I₇₃₂/I₅₃₅) takes place after a certain concentration (7.5 μM) of R. An R-Au NP stock solution is therefore prepared having a final R concentration of 7.5 μM and Au NP concentration of 1 μM. The sensing power of the R-Au NPs is investigated by the use of different concentrations of M and by measuring the absorbance ratio (I₇₃₂/I₅₃₅) vs. concentration of M (Fig. 4). It is apparent from the results that the sensitivity increases as the melamine concentration increases and the sensitivity is much higher even at a M concentration of 0.1 μM (peak ratio = 0.8), which facilitates sensing at a very low concentration of M. The UV-vis kinetics of the optimized R-Au NP solution containing 1.5 μM of M at 30 °C is presented as the inset of Fig. 4. The sensing is detectable from the initial stage and it gradually increases with time, showing a steady state at 8–9 min. This point indicates the completion of the hydrogen bond induced recognition of M turning it into a black solution. The selectivity of the optimized sensor for M is also evaluated by comparing the peak ratio with other molecules e.g.cytosine, uracil, thymine, adenine and guanine under similar conditions (Fig. S4, ESI†). It is noticed from the figure that the selectivity for melamine is ∼10 times higher than any of the above components studied here. Probably the highest sensitivity for melamine comes from the symmetric H-bonding assembly through a larger number of H-bonds with the three R molecules as presented in Scheme 1. As an example the comparison of M and thymine has been made here. R shows H-bond formation with thymine (T)¹⁵ but it is of much lower degree than that of M, as in the RM31 complex 9 H-bonds are formed and in the RT21 complex there are 4 H-bonds causing the M-complex to be stronger than the T-complex (Fig. S5, ESI†). On addition of R-Au NPs, M produces a stronger RM complex than the RT complex making the Au NPs completely free from the R-Au complexes, differing from the case of T. Consequently, the longitudinal plasmon peak at 732 nm (i.e.I₇₃₂/I₅₃₅) is more intense on melamine addition than that of thymine. This selectivity offers the recognition of melamine in foods below the safety level. The efficiency of melamine detection in our system is 0.1 ppm. Compared to other melamine sensing systems our method is significantly important because of the use of Au nanoparticles and riboflavin (a vitamin) [both are biocompatible] and a quick response time with high efficiency.

Fig. 4 Plot of intensity ratio (I₇₃₂/I₅₃₅) vs.melamine concentration with R-Au NPs (Au NPs = 1 μM and R = 7.5 μM). Inset: UV-vis kinetics of the optimized R-Au NP solution containing 1.5 μM of M at 30 °C.

In conclusion, we have developed a biocompatible melamine sensor with very high efficiency and excellent selectivity using riboflavin capped Au nanoparticles.

Acknowledgements

We gratefully acknowledge CSIR, New Delhi (Grant No. 01(2224)/08 EMR-11) for financial support. B. R. and A. S. acknowledge CSIR, New Delhi for providing the fellowship.

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Footnote

† Electronic supplementary information (ESI) available: Figures S1 to S5. See DOI: 10.1039/c0an00599a

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