Supporting Information

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1 Supporting Information Quick-Response agnetic Nanospheres for Rapid, Efficient Capture and Sensitive Detection of Circulating Tumor Cells Cong-Ying Wen a,, Ling-Ling Wu a,, Zhi-Ling Zhang a, Yu-Lin Liu b, Shao-Zhong Wei b, Jiao Hu a, an Tang a, En-Ze Sun a, Yi-Ping Gong b, Jing Yu b, Dai-Wen Pang a a Key Laboratory of Analytical Chemistry for Biology and edicine (inistry of Education), College of Chemistry and olecular Sciences, State Key Laboratory of Virology, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 30072, P. R. China. b Hubei Cancer Hospital, Wuhan, 30079, P. R. China. These authors contributed equally to this work. Corresponding author. dwpang@whu.edu.cn. Phone: ; ax: S

2 S. agnetic Separation Rates of Ns with Different Layers of Nano-γ-e 2 O 3 The magnetic separation rate of Ns was evaluated by their capture efficiency at different attraction times with a commercial magnetic scaffold, which was referred to our previous work. A series of centrifuge tubes containing Ns were put onto the magnetic scaffold for certain times. After that, the suspensions in each tube were drawn out, and their absorbance at 600 nm was measured. As the absorbance at 600 nm was proportional to the N concentration (as shown in igure S), the capture efficiency of Ns at different attraction times could be calculated by the equation: φ t = (-A t /A 0 ) 00% Where φ t is the capture efficiency of Ns at a certain attraction time, A t is the absorbance of suspensions at 600 nm after Ns were attracted for a certain time, and A 0 is the absorbance of the original N solutions at 600 nm. rom igure S2, it can be seen that nearly all Ns were captured at 6 min for Ns with one layer of nano-γ-e 2 O 3,.5 min for Ns with three layers of nano-γ-e 2 O 3, and min for Ns with five, seven, and nine layers of nano-γ-e 2 O 3. igure S. Proportional relationship of absorbance at 600 nm versus N concentration S2

3 igure S2. Capture efficiencies of Ns with one layer (black), three layers (red), five layers (blue), seven layers (green), and nine layers (pink) of nano-γ-e 2 O 3 at different attraction times with a commercial magnetic scaffold. S.2 Recovery of Ns in Whole Blood. Ns in whole blood were re-collected and the recovery of Ns was investigated. A certain amount of Ns were added to whole blood in a centrifuge tube, and after washed 5 times with PBS, the captured Ns and the Ns in the same quantity as the added Ns were respectively dispersed in the same volume of PBS to measure their absorbance at 600 nm. The N recovery was calculated by the equation: Recovery = A c /A 0 00% Where A c is the 600 nm absorbance of Ns captured, and A 0 is the 600 nm absorbance of the Ns in the same quantity as the added Ns. The experiment was performed three times and the recovery was calculated to be (98.±5.6)%. S.3 Confirmation of the Anti-EpCA Antibody on Immunomagnetic Nanospheres (INs) Experiments were designed to prove that mouse anti-epca antibody was successfully S3

As shown in igure S3, the green fluorescence of ITC on the INs was obvious, while no ITC fluorescence was observed on the Ns.

4 conjugated to Ns. INs and Ns respectively reacted with ITC-labeled goat anti-mouse IgG. After 30 min incubation, they were washed 3 times with PBS by magnetic separation, and then observed under a fluorescence microscope. As shown in igure S3, the green fluorescence of ITC on the INs was obvious, while no ITC fluorescence was observed on the Ns. This indicated that mouse anti-epca antibody was successfully conjugated to Ns. igure S3. Bright and fluorescence images of the INs (A, B) and Ns (C, D) after reacted with ITC-labeled goat anti-mouse IgG. S. Estimation of the Amount of Active Affinity Sites on each IN Goat anti-rabbit antibody was taken as a model to modify Ns to estimate the absolute value of active affinity sites on each IN. irst, goat anti-rabbit antibody was conjugated with the Ns with carbodiimide chemistry, and then the obtained anti-rabbit INs were reacted with ITC-labeled rabbit IgG. After incubation for h, the INs captured ITC-labeled rabbit IgG and were washed with PBS, and their fluorescence intensity was then measured by a luorolog-3 fluorescence spectrometer (Horiba Jobin Yvon). Besides, in the presence of Ns S

5 at the same concentration with that of INs, a standard curve (igure S) was got by recording the fluorescence intensities of ITC-labeled rabbit IgG at different concentrations. Based on these, the number of the active affinity sites on each IN could be calculated. The experiment was operated three times, and the number of the active affinity sites on each IN was evaluated to be about 00. igure S. Standard curve of ITC-labeled rabbit IgG of different concentrations in the presence of Ns at the concentration of per ml. S.5 Stability of the INs The bioactivity of INs was monitored over a long period of storage time, which was evaluated by the capture efficiency of INs to tumor cells (igure S5A). It can be seen that INs could capture more than 95% of Hep G2 cells even after month storage. Besides, DLS characterization (igure S5B and C) also showed that hydrodynamic size of INs changed little with increasing storage time, giving 0.8 nm (PDI: 0.29) after four month storage (igure S5C) compared with 05.7 nm (PDI: 0.25) for newly-prepared INs (igure S5B). These results confirmed the stability of the INs. S5

6 igure S5. (A) Capture efficiency of INs to Hep G2 cells at different storage times. (B) Hydrodynamic size of the newly-prepared INs. (C) Hydrodynamic size of the INs after four month storage. S.6 Capture of Tumor Cells with Immunomagnetic luorescent Nanospheres (INs) To demonstrate the binding between immunonanospheres and cells, immunomagnetic fluorescent nanospheres (INs) were used to treat SK-BR-3 cells and Jurkat T cells. The captured cells were observed under a fluorescence microscope, which was shown in igure S6. It can be seen that red fluorescent immunonanospheres successfully bound to the SK-BR-3 cell surface (igure S6A-C), while no cell was captured and only INs were present in the Jurkat T cell control group (igure S6D-). The magnetic fluorescent nanospheres were also prepared with the layer-by-layer assembly method (Three layers of hydrophobic CdSe/ZnS quantum dots, and five layers of nano-γ-e 2 O 3 ). S6

7 igure S6. icroscopic images of the cells captured with INs. Bright field (A), fluorescence field (B), and erge of bright field and fluorescence field (C) of SK-BR-3 cell experimental group. Bright field (D), fluorescence field (E), and erge of bright field and fluorescence field () of Jurkat T cell control group. S.7 Detection of CTCs in Peripheral Blood Samples from Cancer Patients and Healthy People. Table S. Quantification of CTCs of blood samples from patients with epithelial cancers. Sample No. Cancer Type Gender Volume Processed/mL CTCs Colon.5 2 Colon 3 Colon.2 3 Colon Lung.9 3 Lung 2 Lung S7

8 5 Lung 6 Lung Lung Breast.5 9 Breast Table S2. Quantification of CTCs of blood samples from healthy people. Healthy subject Sample NO. Gender Volume processed (ml) CTCs Hu, J.; Xie,.; Wen, C. Y.; Zhang, Z. L.; Xie, H. Y.; Liu, A. A.; Chen, Y. Y.; Zhou, S..; Pang, D. W., A ulticomponent Recognition and Separation System Established via luorescent, agnetic, Dualencoded ultifunctional Bioprobes. Biomaterials 20, 32, S8

Elecrtonic Supplementary Information. Application of quantum dot barcodes prepared using biological self-assembly to multiplexed immunoassays

Elecrtonic Supplementary Information Application of quantum dot barcodes prepared using biological self-assembly to multiplexed immunoassays Sakandar Rauf, Andrew Glidle and Jonathan M Cooper Department