Supporting Information. For. A Dual Electrochemiluminescence Signal System for In-Situ. and Simultaneous Evaluation of Multiple Cell-Surface
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1 Supporting Information For A Dual Electrochemiluminescence Signal System for In-Situ and Simultaneous Evaluation of Multiple Cell-Surface Receptors Bin Zhou, Youyi Qiu,, Qingqing Wen, Mingyao Zhu and Peihui Yang,*, Department of Chemistry, Jinan University Guangzhou , PR China Corresponding author: Peihui Yang, Ph.D, Professor Department of Chemistry, Jinan University Guangzhou , PR China typhjinan@126.com Tel/Fax: S1. Results and discussion S1.1. Characterization of Au@luminol and CdS QDs nanoprobes S-1
2 Transmission electron microscopic (TEM) images were used to characterize the size and the morphology of the obtained and CdS QDs nanoprobes. Figure S1A showed a homogeneous distribution of around 35 nm for Au@luminol in diameter. It was observed that the surface of AuNPs was covered by luminol, which indicated that the Au@luminol was prepared. Figure S1B displayed that the average size of CdS QDs was 7 nm. Besides, absorption spectra showed that Au@luminol had the absorption peak in 530 nm reflects the absorption of AuNPs, and the absorption peaks in 300 nm and 360 nm comes from the absorption of luminol (Figure S1C), suggested that luminol was successfully combined on the surface of AuNPs. Figure S1D exhibited that the absorption peak of CdS QDs was about 370 nm. These results indicated that Au@luminol and CdS QDs had been successfully prepared. Figure S1. TEM image of (A) Au@luminol and (B) CdS QDs; Absorption spectra of (C) Au@luminol and (D) CdS QDs in aqueous solution. S1.2. Optimization of the proposed cytosensor The ECL performance of the proposed cytosensor was influenced by many factors. To obtain high sensitivity, some experiment parameters were investigated. The concentration of luminol had an important influence on the ECL intensity of the prepared Au@luminol nanoprobe. As shown in Figure S1A, the intensity increased obviously with the concentration of luminol increasing to 0.2 S-2
3 M and then tended to level off, indicating a saturated concentration of luminol on the surface of AuNPs. Thus, the optimum concentration of luminol was chosen as 0.2 M. The recognition ability and the sensitivity of the nanoprobes were determined by the amount of Au@luminol and CdS QDs immobilized on each nanoprobe, respectively. Increasing the amount of Au@luminol or CdS QDs on each probe would enhance the ECL intensity. As shown in Figure S1B and S1C, the maximum ECL emissions were achieved at the volume ratio of Au@luminol to ConA was 30:1 and CdS QDs to EGF was 20:1, respectively. The MCF-7 cells and sensing interface incubation time was another important experimental parameter. As the cell capture time (10 4 cells ml -1 of MCF-7 cells) progressively increased, the current signal for cytosensor in 0.1 M PBS (ph 7.4) containing 5.0 mm [Fe(CN) 6 ] 4- / 3- and 0.1 M KCl decreased after 20 min and then tended to level off, indicated that the optimum capture time was at 20 min (Fig. S1D). When incubating the captured cells with Au@luminol-ConA and CdS QDs-EGF nanoprobes, the maximum ECL intensity were obtained both after incubation for 20 min, as shown in Figure S1E and S1F. According to the results in Figure S1, these optimal experiment parameters were used in subsequent measurements. Figure S2. ECL intensity of Au@luminol obtained with different luminol concentrations (A); ECL intensity of the volume ratio of Au@luminol to ConA (B) and CdS QDs to EGF (C); dependence of current signal for cytosensor in 0.1 M PBS (ph 7.4) containing 5.0 mm [Fe(CN) 6 ] 4- / 3- and 0.1 M KCl on the capture time of MCF-7 cells (D); dependence of ECL intensity for Au@luminol-ConA (E), CdS QDs-EGF (F) on the recognition time of MCF-7 cells. S-3
4 S1.3. Evaluation of cross-reactivity To evaluate the cross-reactivity of the multiplex ECL cytosensor, three control tests were carried out as follows: MCF-7 cells was captured on the MUC1 aptamer-functionalized sensing interface, and then (1) single detection of mannose was performed by using 10, 20, 30, 40, 50, 60, 70, 80 µl Au@luminol-ConA nanoprobe, (2) single detection of EGFR was performed by using 10, 20, 30, 40, 50, 60, 70, 80 µl CdS QDs-EGF, (3) mannose and EGFR were simultaneously detected by using Au@luminol-ConA and CdS QDs-EGF. The ECL intensity increased obviously with the dosage of each nanoprobe increasing to 60 µl and then tended to level off in Figure S3A-D. Meanwhile, two potential-resolved ECL signals increased continuously and kept at a steady state finally (Figure S3E and S3F). The results showed that the single detection of one receptor was good agreement with the simultaneous detection of two receptors, indicating that the cross reaction between the two nanoprobes had not been observed in our research. S-4
5 Figure S3. ECL behaviors of (A) CdS QDs-EGF, (C) and (E) and CdS QDs-EGF with different dosages in 0.1 M PBS (ph 7.4) containing 0.1 M K 2 S 2 O 8 ; relationship curves between ECL intensity and dosage of (B) CdS QDs-EGF, (D) Au@luminol-ConA, (F) Au@luminol-ConA and CdS QDs-EGF. S1.4. Selectivity, reproducibility and stability of the multiplex cytosensor To evaluate selectivity, reproducibility and stability, the multiplex cytosensor was investigated. MCF-7 cells with high receptors expression and human embryonic kidney (HEK) cells with low receptors expression were used to study the selectivity of the cytosensor. Figure S4A showed that two significant ECL signals were observed in the presence of MCF-7 cells, but S-5
6 the signals of HEK cells were weak. Compared to MCF-7 cells, ECL intensity of the mixture of MCF-7 cells and HEK cells did not show a clear change, which exhibited that the multiplex cytosensor possessed good selectivity (Figure S4A). In addition, six parallelly prepared electrodes were incubated with the solution containing cells ml -1 of MCF-7 cells to illustrate reproducibility. The relative standard deviation (RSD) was 4.2 % for Au@luminol-ConA nanoprobe and 3.9 % for CdS QDs-EGF nanoprobe, respectively, indicating good reproducibility of the ECL cytosensor (data not shown). The stability of the cytosensor was evaluated under consecutive cyclic potential scans for 10 cycles at cells ml -1 of MCF-7 cells. No significant ECL peak signal change was observed in Figure S3B. Furthermore, the cytosensor was stored at 4 o C for more than two weeks and the response was recorded periodically. Only a small difference in the ECL signals response (less than 10%) was found even at the fourteenth day (data not shown). The results indicated that the proposed cytosensor had satisfying selectivity, reproducibility and stability. Figure S4. (A) Selectivity of these nanoprobes after incubation with different kinds of cells; (B) stability of ECL intensity of proposed cytosensor as time increases. S-6
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