Haiyan Ma, Zongbing Li, Ning Xue, Zhiyuan Cheng, Xiangmin Miao*

Electronic Supplementary Material

A gold nanoparticle based fluorescent probe for simultaneous recognition of single-stranded DNA and double-stranded DNA

School of Life Science, Jiangsu Normal University, Xuzhou 221116, PR China

*Corresponding author. Tel.: +86 516 83403170

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Preparation of AuNPs

AuNPs are prepared according to literature method [1]: Briefly, 20 mL of HAuCl4 (1 mM) solution was heated and stirred simultaneously until boiling. Then, 2.2 mL of sodium citrate (38.8 mM) was added into the boiling HAuCl4 (1 mM) solution and kept stirring consistently for another 5 min until the color of the mixed solution turned from light yellow to wine red and without changing within 15 min. After cooling to room temperature, the solution was stored in a refrigerator at 4°C for further uses. All of the glass-wares that used for the preparation of AuNPs solution were washed thoroughly with aquaregia prior to use. The average size of such AuNPs estimated from TEM images were about 16 nm (Fig. S1A). Meantime, the zeta potential of such AuNPs monitored from dynamic light scattering (DLS) were negatively charged (Fig. S1B).

Fig. S1TEM images (A) and (B) the zeta potential analysis of AuNPs.

Fig.S2 Excitation (a) and emission (b) spectra for fluorescent DNA probe: (A) FAM labeled P1, and (B) ROX labeled P2.

Fig. S3 (A) Emission spectra of FAM labeled P1 with the excitation wavelength (from 340 to 520 nm). (B) Emission spectra of ROX labeled P2 with the excitation wavelength (from 340 to 640 nm). All of the spectra were recorded at room temperature.

Optimization of the experimental conditions

The concentrations of P1 and P2 play important roles in the performance of the sensor. Thus, the effects of P1 and P2 concentrations were investigated in the presence of 300 nM of T1 and Ta/Tb, respectively. As shown in Fig.S4A, the fluorescence signal of P1 increased dramatically along with the increase of P1 concentration over the range of 10-500 nM, then reached a plateau. Thus, 500 nM of P1 was selected for the experiments. Meantime, it could be seen from Fig. S4B that the increase in P2concentration from 10 to 500 nMdirectly resulted in the increase in the fluorescence signal of P2. Consequently, 500 nM of P2 was selected for the experiments.

The concentration of spermine is a key factor to improve the stability of triplex DNA structure [2]. Fig. S4C showed that the fluorescence signal of P2 increased obviously with the concentration of spermine ranging from 1 to 15 μM, which illustrated that 15 μM of spermine was enough for the formation of stabled triplex DNA. Therefore, 15 μM of spermine was employed in this work.

Fig. S4Effect of the concentration of P1 (A) and P2 (B) on the response of the sensing system in the presence of 300 nM ssDNA and dsDNA, respectively; (C) Effect of the concentration of spermine on the response of the sensor in the presence of 300 nM dsDNA. The error bars indicated the standard deviations of three replicates.

Fig. S5 (A) Fluorescence spectra of P1’ and P2’ upon the addition of different concentrations mecA gene and simian virus 40 sequences simultaneously; (B) and (C) were the linear plot of the assay for mecA gene and simian virus 40 sequencesdetection. The error bars indicated the standard deviations of three replicates.

Table S1Addition and Recovery experiments of ssDNA in human serum samples

Serum samples(ssDNA) / Added / Found / Recovery (%) / RSD (%)
1 / 1.0 (nM) / 1.01(nM) / 101.6 / 3.45
2 / 300 (nM) / 293.21 (nM) / 97.7 / 4.12
3 / 500 (nM) / 494.73 (nM) / 98.5 / 3.85

Table S2Addition and Recovery experiments of dsDNA in human serum samples

Serum samples(dsDNA) / Added / Found / Recovery (%) / RSD (%)
1 / 1.0 (nM) / 0.99(nM) / 98.7 / 4.57
2 / 300 (nM) / 296.91 (nM) / 99.1 / 2.18
3 / 500 (nM) / 506.46 (nM) / 101.4 / 3.12

Reference

1. Grabar K, Freeman R, Hommer M, Natan M(1995)Preparation and characterization of Au colloid monolayers.AnalChem67(4): 735-737.

2. Thomas T, Thomas T (1993) Selectivity of polyamines in triplex DNA stabilization. Biochemistry 32(50): 14068-14074.