Electronic Supporting Material on the Microchimica Acta publication entitled
Gold nanoparticle-based colorimetric ELISA for quantification of ractopamine
Shuaijuan Han, Tianjiao Zhou, Bingjie Yin, and Pingli He*
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, P. R. China
*Corresponding author. State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing, 100193, China
Tel: 86-10-62733588
E-mail address: .
Optimization of AuNPs growth
1.Optimization of concentration of HAuCl4
The first parameter optimized was concentration of the HAuCl4 solution. Four concentrations of HAuCl4 (0.1, 0.2, 0.4, 1.0 mM) were tested with 100μM hydrogen peroxide. When the HAuCl4 concentration exceeds 1 mM, a light yellow precipitate can be observed after the reaction, which indicates conglomeration of the AuNPs. However, when the HAuCl4 solution concentration was below 0.1 mM, the colorimetric assay suffered from reduced sensitivity. When the HAuCl4 solution concentration was 0.2 mM, the absorbance at 560 nm was maximized (Figure S1).
SFig. 1. Influence of concentration of the HAuCl4 on generation of gold nanoparticle: (A) Photograph showed the AuNPs solutions with different colors and intensities after 15 min. (B)The corresponding absorbance values at 560 nmfor different HAuCl4concentrations of (n=3).
2. Optimization of incubation time and pH of the buffer solution
The effects of incubation time and pH of the buffer solution on AuNPs growth also were tested. For the optimization of them, we just fix one parameter to optimize another parameter. For example, when the buffer was set at pH 6, the different incubation time (5, 10, 15, 20 min) were observed.FigureS2. shows absorbance valueat 560 nm of the AuNPs after mixing of hydrogen peroxide with HAuCl4 under different incubation time and pH buffer. The maximum absorbance was nearly reached at about 15 min of incubation. Further increase the incubation time, the blank also began to become blue.According to the rapidity of detection and lower background interface, the optimal incubation time was determined to be 15 min. Weak acidic buffer favors the growth of AuNPs, strongly acidic or basic conditions (pH 4 and pH 9) yielded no AuNPs formation and weak absorbance at 560 nm. The optimal pH condition was 6.0 because the absorbance at 560 nm was maximized.
SFig. 2. Influence of buffer pH and incubation time on the absorbance measured at 560 nm with adding the hydrogen peroxide to the HAuCl4 solution. Incubation time:Line A. 5 min, Line B. 10 min, Line C. 15 min, Line D. 20min (n=3).
3Optimization ofconcentration of gold seeds
To further enhance sensitivity, the gold nanoparticle seeds were added into the reaction system.Sevenvolumes of gold seeds (0, 5, 10, 15, 20, 25, 30L) were tested. The results show that addition of 5 L gold seeds enhanced the speed and sensitivity of the reaction (Figure S3).
SFig. 3. Influence of the amounts of gold seeds on generation of gold nanoparticle: (A) Photograph showed the AuNPs solutions with different colors and intensities after 15 min. (B)The corresponding absorbance values at 560nmfor different amount of gold seeds (0, 5, 10, 15, 20, 25, 30L) (n=3).
4. Interferences
As acomparison, we tested the possibility of other drugsto interfere with the detection of ractopamine (Fig. S4).
Fig.S4. Visual color change of AuNPs upon addition of 100 g·mL-1of each drug under optimized conditions (from left to right: Ractopamine, clenbuterol,salbutamol,sulfadiazine, sulfamerazine).