Electronic supplementary material

Competitive immunoassay for imidaclothiz using upconversion nanoparticles and gold nanoparticles as labels

Hongjie You•Xiude Hua*•Lu Feng•Nana Sun•Qi Rui•Limin Wang•Minghua Wang*

*Corresponding authors, e-mail: (M. Wang), (X. Hua)

Optimization of method

The experimental parameters, including the concentration of imidaclothiz (IMI) antigen conjugated AuNPs,pH value, ionic strength, incubation time and organic solvent, were sequentially studied to determine the optimal conditions. TheIFE-based competitive immunoassay was performed with the concentrations of IMI antigen conjugated AuNPs from 0 nmol L-1 to 2.0 nmol L-1. And then, the IFE-based competitive immunoassay was run in sodium borate buffers with various pH values (from 5 to 9),concentrations ofNa+ (from 0.02 mol L-1 to 0.16 mol L-1),incubation times (from 10 min to 70 min)and concentrations ofmethanol (from 0 to 10%). The evaluation was based on the fluorescence recovery ((I-I0)/ I0, I and I0 represent the fluorescence intensities at 544 nm with and without 1000 ngmL−1 of IMI, respectively) and the high recovery was the most desirable.

Fig.1B shows the variety of the fluorescence quenching with increasing the concentration of antigen conjugated AuNPs. When the concentration of antigen conjugated AuNPs reach to 1.6 nmol L-1, the fuorescence is quenched remarkably. To avoid the interference of excessive AuNPs, 1.6 nmol L-1 antigen-conjugated AuNPs was selected.The IFE-based competitive immunoassay is based on the antigen-antibody interactions, ionic strength, pH value and incubation time are additional factors influencing the sensitivity of the assay. The pH 8.0, 0.12 mol L-1 Na+ and incubation for 50 min areselected because the immunoassay shows the highest fluorescence responses (Fig. S3A,S3B and S3C). The organic solvents is necessary for sample pretreatment and dissolution of analyte, but high concentration of organic solvent often affects antigen-antibody binding. Methanol is commonly used as an organic co-solvent in immunoassays because of its weaker effect on antigen-antibody interaction. When the final concentration of methanol is less than or equal to 5% in reaction solution, the effects of methanol on the IFE-based competitive immunoassayare negligible (Fig. S3D). In conclusion, the optimum parameters of the IFE-based competitive immunoassay are1.6 nmol L-1 antigen-conjugated AuNPs,pH 8, ionic strength of 0.12mol L-1, incubation for 50 min and5% methanol.

Table S1 Thebackground fluorescence of different matrices by using IFE-based competitiveimmunoassay and FPIA

Method / Different matrix background fluorescence values
Paddy water / Soil / Pear / Rice / Apple / Tomato / Pakchoi / Cabbage
IFE / -0.172 / -0.532 / -0.499 / -0.061 / -0.109 / -0.233 / -0.213 / -0.075
FPIA / 114.95 / 172.54 / 874.08 / 529.44 / 374.84 / 324.54 / 241.04 / 124.29

Table S2Results of the IFE-based competitiveimmunoassay and HPLC for IMI residues in real sample

Matrix / Method / Sample (ng mL−1 or ng g−1) / Significance test (P)
1 / 2 / 3 / 4 / 5 / 6
Paddy water / HPLC / 163.1 / 129.4 / 95.6 / 74.1 / 49.0 / 9.4 / 0.8171
IFE / 142.1 / 121.6 / 91.8 / 63.2 / 42.1 / 10.2
Pear / HPLC / 126.5 / 79.7 / 62.0 / 58.7 / 38.5 / 23.3 / 0.9219
IFE / 119.4 / 87.2 / 65.8 / 55.4 / 35.7 / 26.1

Fig.S1 TEM images of NaYF4:Yb,ErUCNPs (A), AuNPs (B), amino-modified NaYF4:Yb,ErUCNPs (C), and X-ray diffraction spectrogram of NaYF4:Yb,ErUCNPs (D)

Fig. S2The fourier transform infrared spectroscopy of amino-modified NaYF4:Yb,Er UCNPs. The peaks in the region around 3382 cm-1 and 1633 cm-1 are assigned to the hydroxyl stretching vibration and bending vibration bands of the amine group (-NH2). A strong absorption at 1089 cm-1 is attributed to the stretching vibration of silicon-oxygen bond (Si-O). The peaks at 2988 cm-1 and 2890 cm-1 are belong to the asymmetric and symmetricstretchingvibrations of the methylene group, respectively, which exists in the hydrolysis product of APTES. The peaks at 3382 cm-1, 2988 cm-1, 2890 cm-1 and 1633 cm-1 together verify the silica-coated UCNPs have been modified with amino groups

Fig. S3 Effect of pH (A), concentration ofNa+ (B), incubation time (C) and concentration of methanol (D)on IFE-based competitive immunoassay

Fig. S4 Matrix interference onIFE-based competitive immunoassay. Standard curves for IMI in the buffer, soil (A), pear(B), rice(C), cabbage(D), tomato(E), apple(F), pakchoi (G) matrices using IFE-based competitiveimmunoassay