Study on the Interaction Among Pyronine Y, Potassium Bromate and Naphthols By

Electronic Supplementary Material

Determination of metallothioneins based on the enhanced peroxidase-like activity of mercury-coated gold nanoparticles aggregated by metallothioneins Xue-Jiao Li a, Yong-Sheng Wang a*, Sheng-Yuan Yang a, Xian Tang a, Lu Liu b, Bin Zhou a, Xiao-Feng Wang a, Yu-Feng Zhu a, Yang-Qin Huang a, Shun-Zhen He a a College of Public Health, University of South China, Hengyang 421001, PR China b College of Traditional Chinese Medicine, Hunan University of Chinese Medicine, Changsha 410208, PR China

1. Procedures for measuring XPS, FTIR spectra and ICP-MS

1.1. X-ray photoelectron spectroscopy (XPS) measurement X-ray photoelectron spectroscopy (XPS) was performed using a Thermo Scientific Escalab 250Xi X-ray photoelectron spectrometer (Waltham, UK) with a monochromated Al K alpha X-ray source (1468.6ev). The solutions of AuNPs, AuNP-Hg(II) and AuNP-Hg(II)-MTs were prepared respectively. The residues obtained by c entrifug ing were coated onto the face of prepared 1×1 cm glass slides, which were soaked in 10 % nitric acid overnight, and diped in the 30 % H2O2 for another 8 hours. Next, all the samples were taken into the vacuum drying oven for drying before measurement.

1.2. Inductively coupled plasma mass spectrometry (ICP-MS) measurements The amounts of Hg(II) atoms/ions on the AuNP surfaces were determined by Agilent Technologies 7700 series ICP-MS (California, USA). The solutions of AuNPs, AuNP-Hg(II) and AuNP-Hg(II)-MTs were prepared respectively, and the supernatant fluid obtained by c entrifug ing was measured each for three times. The operating parameters of ICP-MS instrument were as follows: the temperature of atomizing chamber is 2 °C, RF power 1550 W, carry gas flow 1L min–1. 1.3. Fourier-Transform Infrared (FTIR) spectrum measurement FTIR spectra were measured on a Shimadzu IRPrestige-21 FTIR spectrometer (Kyoto, Japan), equipped with a KBr beam splitter, a standard source and a DTGS Peltier-cooled detector. The solutions of AuNPs, AuNP-Hg(II) and AuNP-Hg(II)-MTs were prepared respectively. Thereafter, the resulting solutions were drawed onto the middle of two KBr pellet with capillary tube. The KBr pellet spectra of samples were obtained in the range from 4000 to 400 cm–1 with a resolution of 4 cm−1.

2. Choice of Materials

Fig. S1. Effect of the concentration of AuNPs on the assay system.

cHg(II) = 10.0 µM, cMTs = 61.6 nM, cABTS = 0.50 mM, cH2O2 = 1.2 mM.

Fig. S2. Effect of the concentration of Hg(II) on the assay system. cMTs = 61.6 nM, cAuNPs = 6.46 nM, cABTS = 0.50 mM, cH2O2= 1.2 mM.

Fig. S3. Effect of the concentration of ABTS on the assay system.

cHg(II) = 8.0 μM, cAuNPs = 6.46 nM , cMTs = 61.6 nM, cH2O2 = 1.2 mM.

Fig. S4. Effect of the concentration of H2O2 on the assay system.

cHg(II) = 8.0 μM, cAuNPs= 6.46 nM, cMTs = 61.6 nM, cH2O2 = 1.2 mM. 3. Mechanism on the interaction among MTs, AuNPs and Hg

Fig. S5. Effect of adding sequence for reagents

1. HAc-NaAc, AuNPs, MTs, HgCl2, ABTS, H2O2; 2. HAc-NaAc, AuNPs, HgCl2, MTs,

ABTS, H2O2; 3. HAc-NaAc, HgCl2, AuNPs, MTs, ABTS, H2O2; 4. HAc-NaAc, AuNPs,

MTs, HgCl2, H2O2, ABTS; 5. HAc-NaAc, MTs, AuNPs, HgCl2, H2O2, ABTS; 6. HAc-

NaAc, MTs, AuNPs, HgCl2, ABTS, H2O2; 7. HAc-NaAc, ABTS, H2O2, AuNPs, MTs,

HgCl2; 8. ABTS, H2O2, HAc-NaAc, AuNPs, MTs, HgCl2.

Fig. S6. Visual observation (A) and UV-Vis spectra (B) for AuNPs- Hg(II)-MTs system.

a. AuNPs, b. AuNPs+Hg(II), c. AuNPs+MTs, d. AuNPs+MTs+ Hg(II), cAuNPs = 3.25 nM; cHg(II)

= 16.7 μM; cMTs = 0.26 μM. Fig. S7. TEM image for AuNPs-Hg(II)-MTs system.

A. AuNPs; B. AuNPs+Hg(II); C. AuNPs+MTs; D. AuNPs+MTs+Hg(II); cAuNPs= 3.25 nM, cHg(II) = 16.7 µM, cMTs = 0.26 µM. Fig. S8. Hydrodynamic diameter distribution plots as determined by dynamic light scattering (DLS) measurements. A. AuNPs; B. AuNPs+Hg(II); C. AuNPs+MTs; D. AuNPs+ MTs+

Hg(II); cHg(II) = 3.25 nM, cAuNPs= 16.7 µM, cMTs = 0.26 µM.

Fig. S9. Effect of pH on the absorbance of assay system.

cHg(II) = 10.0 μM, cMTs = 61.6 nM, cAuNPs= 8.07 nM, cABTS =0.5 mM, cH2O2 = 1.2 mM.

4. Sensitivity of the MTs sensing system

Fig. S10. Plot of visual observation for MTs assay by a peroxidase mimic.

2+ 2+ 2+ 1. MTs + Hg + ABTS + H2O2, 2. AuNPs + Hg + ABTS + H2O2, 3-5. AuNPs + Hg + MTs + ABTS + H2O2, cHg(II) = 5.0 μM, cAuNPs = 9.75 nM, cABTS = 0.5 mM, cH2O2 = 1.2 mM, cMTs (μM)/(3- 5) = 0.0, 0.0616, 0.154, 0.264. 5. Selectivity of the MTs-sensing system

Fig. S11. Response of the MTs sensing system to various metal ions.

cHg(II) = 8.0 μM, cMTs = 61.6 nM, cAuNPs = 8.07 nM, cABTS = 0.7 mM, cH2O2 = 1.2 mM, ck(I), Na(I), Mn(II) + = 40 μM, cZn(II), Fe(II), Al(III), Mg(II), citric acid = 4.0 μM, cPb(II) = 8.0 μM, cNH4 = 1.232 μM, cethanol, sodium oxalate

= 123.2 μM, cL-cysteine = 616 nM, ccarbamide, glucose, Ca(II) = 61.6 μM. Blank, in the absence of potentially interfering substances

Table S1. Comparison of this method with other strategies for MTs assay a

Methods Linearity ranges LOD Samples Recovery Refs. (nM) (nM) (%) Col 25.6-308 7.67 Human urine 94.8–103.9 22 CV - 120 Human Blood - 31 HPLC-AAS - 70.8 Rat liver 103 32 UV 259-1540 776 Human urine 91.8 33 Flu 10.3-1230 3.1 Human urine 96.43–103.0 34 RLS 256-1540 76.8 Human urine 93.34–95.13 34 AuNPs-Hg-MTs 4.34-49.3 1.3 Human urine 100.2–104.8 This work a Col: colorimetric method; CV: cyclic voltammetry; HPLC-AAS: high-performance liquid chromatography-atomic absorption spectrometry; UV: ultraviolet spectrophotometry; Flu: fluorometric method; RLS: resonance light scattering. References [31] Petrlova J, Potesil D, Mikelova R, Blastik O, Adam V, Trnkova L, Jelen F, Prusa R, Kukacka J, Kizek R (2006) Attomole voltammetric determination of metallothionein. Electrochim Acta 51: 5112-5119 [32] Nostelbacher K, Kirchgessner M, Stangl GI (2000) Separation and quantitation of metallothionein isoforms from liver of untreated rats by ion-exchange high-performance liquid chromatography and atomic absorption spectrometry. J Chromatogr B 744:273-282 [33] Liu L, Li Q, Xue JH, Zhou B, Wang YS (2012) Determination of metallothioneins by ultraviolet spectrophotometry with ciprofloxacin-Cu complex. Appl Chem Ind 41:507-509 [34] Liu L, Wang YS, Xue JH, Yang HX, Li Q, Zhou B, Wang JC, Yin JC, Wang YS, Xiao XL (2013) Determination of metallothioneins by fluorescence and resonance light scattering strategies based on ciprofloxacin–Cu(II) system. J Lumin 138:251-257