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Antibody-free colorimetric determination of total aflatoxins by mercury(II)- mediated aggregation of lysine-functionalized gold nanoparticles

Bibai Du1,a, Peilong Wang2,a, Can Xiao1, Ying Zhou1, Li Wu1,3, Hong Zhao1, Xiaoou Su2, Jingkui

Yang1,*, Yujian He1,3,*

1School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences,

Beijing 100049, China

2Key Laboratory of Agro-Product Safety and Quality, Ministry of Agriculture, Institute of Quality

Standards & Testing Technology for Agro-Products, Chinese Academy of Agricultural Sciences,

Beijing 100081, China

3State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China ESI-MS condition for the Identification of AFs-Hg2+complexes

The mass spectrometric measurements were recorded using a Xevo TQ tandem MS instrument (Waters, USA) fitted with electrospray ionization (ESI) source. The solutions of the individual aflatoxin (1.5 ppm) and mixture of aflatoxin (1.5 ppm) and Hg2+ (10-4 M) were directly infused into the ESI source via the instrument syringe pump at a ﬂow rate of 5 μL/min. Spectra were acquired over a mass range of m/z 100–2000. Experimental conditions were ion modes, capillary voltage of 2800 V, cone voltage of 20-100 V, ion source temperature of 150 °C, desolvation gas temperature of 450 °C, desolvation gas flow rate of 800 L/h. Other instrumental parameters were automatically adjusted to optimize the signal-to-noise ratio.

2+ Fig.S1. Selected regions of full scan positive ion electrospray mass spectra of (a) AFB1 (b) AFB1-Hg complexes

2+ 2+ 2+ (c) AFB2 (d) AFB2-Hg complexes (e) AFG1 (f) AFG1-Hg complexes (g) AFG2 (h) AFG2-Hg complexes. All aflatoxins with addition of Hg2+ showed prominent m/z peaks corresponding to the occurrence of dimeric AFs-

Hg-AFs species. The peaks at m/z 825, m/z 829, m/z 857, and m/z 861 correspond to the dimer AFB1-Hg-AFB1,

2 AFB2-Hg-AFB2, AFG1-Hg-AFG1, and AFG2-Hg-AFG2, respectively. The results indicated that all the four kinds of aflatoxins can coordinate with Hg2+ to form AFs-Hg2+-AFs complexes.

Theoretical calculation on the AFs-Hg2+-AFs complexes

All calculations were performed using the Gaussian 09 Programs [1]. The geometries were fully optimized using the density functional theory (DFT) Becke three-parameter Lee-Yang-Parr (B3LYP) [2-4] method. Each structure was classified as a minimum (no imaginary frequencies) by frequency analysis calculations at the B3LYP/6-31G (d,p) level. The energies were corrected with zero-point energies (ZPEs) calculated at the B3LYP/6-311+G (d,p) level.

Fig. S2. DFT-optimized model of AFs and Hg2+ (2:1). AFs bound to Hg2+ through two carbonyl oxygen atoms and Hg2+ had tetrahedral coordination configuration. The distances of Hg2+ from the two oxygen atoms were 2.19 Å 2+ 2+ and 2.35 Å in the structures of AFB1-Hg complexes and AFB2-Hg complexes, and 2.21 Å and 2.25 Å in the 2+ 2+ structures of AFG1-Hg complexes and AFG2-Hg complexes. Optimization of experimental conditions

(a) The size of the AuNPs

The sensitivities of AuNP-based sensors usually vary with the size of AuNPs [5,6]. AuNPs in the size range from 13-50 nm was prepared using different C6H5Na3O7/HAuCl4 ratios according to a previous literature [7]. After AuNPs of different size were modified with lysine, Hg2+ ions at same concentration were added into the Lys-AuNPs suspensions. The experimental results (Fig. S3) show that AuNPs with particle diameter of 25 nm and 36 nm exhibits similar sensing response which is the best. However, AuNPs with size of 13 nm show lower response to Hg2+ ions due to the reduced surface area of smaller AuNPs, which limits accommodation of the ligand lysine by the AuNPs [5]. When using AuNPs with larger diameter in 50 nm, AuNPs demonstrated a light purple color after the modification with lysine, indicating AuNPs aggregates to some extent, which is not suitable for further experiment. Thus, AuNPs with diameter of 25 nm was selected as the best size.

Fig. S3. Effect of particle size on the aggregation of Lys-AuNPs in the presence of Hg2+ (0.75 μM).

4 (b) Effect of phosphate buffer

To understand whether salt induces the aggregation of Lys-AuNPs, zeta potential measurement of Lys-AuNPs in presence of different concentration of phosphate buffer was performed. As shown in Fig. S4, the absolute value of zeta potential of Lys-AuNPs suspension was higher than 30 mV even in presence of 7.5 mM phosphate buffer, indicating the particles are stable in low concentration of salt [8]. But zeta potential of Lys-AuNPs was 24.7 mV in presence of 10 mM phosphate buffer, showing Lys-AuNPs slightly aggregate in high ionic strength. Therefore, try to avoid the possible negative effect of salt, the colorimetric detection process was carried out without addition of buffer. Fig. S4. Zeta potential of Lys-AuNPs in the presence of different concentration of phosphate buffer (pH=7). (a) 0; (b) 1 mM; (c) 2.5 mM; (c) 5 mM; (d) 7.5 mM; (e) 10 mM.

(c) Concentration of lysine

The concentration of surface ligand will significantly influence the aggregation extent of AuNPs induced by Hg2+ [9,10]. So the concentration of surface ligand lysine was optimized in the assay. As mentioned above, the aggregated AuNPs had two absorption bands centered at 525 nm and 725 nm, so the ratio of absorbance at 725 nm and 525 nm (A 725/A525) was used to evaluate the aggregation degree of AuNPs. A higher A725/A525 value corresponded to the stronger aggregation of AuNPs, while a lower one referred to the better dispersion of AuNPs. The concentration of lysine was optimized from 0 to 2.1 mM, the plot of A725/A525 value versus lysine concentration for AuNPs suspension in the presence of 0.75 μM Hg2+ is shown in Supplementary material (Fig. S5). The

6 A725/A525 value increased sharply in the concentration of lysine range from 0.3 mM to 0.9 mM and reached a plateau when the concentration is 1.2 mM. Thus, 1.5 mM lysine that ensured aggregation of AuNPs was strong enough in the presence of Hg2+ was selected in our experiment.

Fig. S5. Effect of modified concentration of lysine on the aggregation of AuNPs in the presence of Hg2+ (0.75 μM).

(d) Concentration of Hg2+

To achieve the best sensing performance, the concentration of Hg2+ was further optimized. Fig. S6 shows photographic images of Lys-AuNPs in the presence of Hg2+ at different concentrations. It is evident that Hg2+ does not induce aggregation of AuNPs if the concentration of Hg2+ drops to below 0.6 μM. On the other hand, aggregation reaches a maximum if the concentration is increased to 0.75 μM or higher. This is accompanied by a color change from red to gray. However, by decreasing the amount of added Hg2+ ions, sensitivity of the method will be increased. In other words, high concentrations of Hg2+ consume more AFs and have an adverse effect on sensitivity. Therefore, the concentration of Hg2+ at 0.75 μM was chosen in our study.

Fig. S6. The aggregation of AuNPs using lysine (1.5 mM) as capping ligand induced by Hg2+ of different concentrations.

(e) Incubation time

The effect of incubation time on the colorimetric response was also investigated. Fig. S7 shows the anti-aggregation kinetics behavior of Lys-AuNPs by AFs at different concentrations. When the concentration of AFs was lower than 30 ppb, the A725/A525 value of Lys-AuNPs increased sharply with an increase of incubation time and almost attained maximum within 4.5 min. This is because 8 excessive Hg2+ ions existed in the suspension, which bound to Lys-AuNPs and resulted in cross- linking of Lys-AuNPs in short time. When the concentration of AFs was higher than 150 ppb, the

A725/A525 value increased gradually with time and varied slightly after 3 min. This is explained by the fact that most Hg2+ ions have been consumed by AFs to form AFs-Hg2+ complexes. The lack of Hg2+ ions leads to aggregation of Lys-AuNPs. The results show that the de-aggregation of Lys- AuNPs induced by AFs is complete within 5 min. It further reveals that the AFs-Hg2+ complexes are stable and their formation is irreversible. Considering saving the measurement time, the data were recorded at 5 min in subsequent experiments.

2+ Fig. S7. Absorption ratio A725/A525 of Lys-AuNPs containing 0.75 μM Hg versus time at different concentration of AFs. (the data were recorded at the interval of 1.5 min).

Fig. S8. Photographic images of Lys-AuNPs in the presence of different metal ions and AFs. Analysis of AFs by HPLC-FLD

HPLC condition: Chromatographic separation was performed with a 1200 HPLC system (Agilent, USA) consisting of a quaternary pump, an auto sampler, a degasser, a temperature-stable column compartment, an online post-column derivatization device, and a fluorescence detector (FLD). A reverse phase Symmetry C18 column (250 mm×4.6 mm, 5μm particle size, Waters, USA) was used for chromatographic separation and the column temperature was set at 60 °C. The mobile phase was methanol–water (45:55, v/v), at a flow rate of 0.8 mL/min. The derivatization reagent was iodine solution (0.05%) and the temperature of the post-column reactor was 70 °C. The injection volume into HPLC system for both standard and sample was 100 μL. The ﬂuorescence detector was set to excitation and emission wavelengths of 360 and 430 nm, respectively. Each chromatographic run was 15 min.

Fig. S9. HPLC chromatograms of (a) AFs standard solution (each at 5 ppb), (b), rice sample 1, (c) rice sample 2, and (d) rice sample 3. Retention times were around 11.9, 10.4, 9.2 and 8.3 min for AFB1, AFB2, AFG1 and AFG2, respectively. No peak was present in the chromatograms of rice samples, indicating that the selected rice samples for addition and recovery experiments were aflatoxin-free.

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