## 3 Results
### Probing NTERA-2 and CCD-18 Co Cells with **Tmc** Nanocomplex  
As presented in Table 1, the CE index, which reports the conjugation efficiency of TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\) nanorods with mAb against CD133 and PD-L1, was high, ranging from 60% to 100%.  
To evaluate the binding ability of **TMC** to cells, cells were observed under a fluorescence microscope. According to [PERSON] et al., NTERA-2 highly express CD133, so this cell line was chosen for this experiment [11]. As shown in Figure 5, NTERA-2 cells labeled with luminescent nanocomplex **TMC** expressed strong luminescence under fluorescence microscopy compared with the negative control (Figure 5). Although TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\) could bond with the cells, the bonds were weak; therefore, the fluorescence image was not bright.  
The flow cytometry results also provided the percentage of probed cells using the **TMC** nanocomplex. CD133-FITC (Invitrogen; Carlsbad, CA, USA) served as a reference control. As shown in Figure 6, CD133 expression was found in about 95.83% \(\pm\) 7.31% of NTERA-2 cells when stained with CD133-FITC. A similar result (97.77% \(\pm\) 5.69%) was found in NTERA-2 cells that were incubated with **TMC** (\(p\) > 0.05). The percentage of CD133-positive cells was only 1.11% \(\pm\) 0.06% for NTERA-2 cells incubated with unconjugated TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O nanorods.  
CCD-18 Co cells were also incubated with the nanorods conjugated with mAb under the same conditions. However, luminescent expression of CCD-18 Co cells bound with nanomaterials was not  
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**No.** & **Samples** & **Input mAb** & **Free mAb in Supernatant** & **CE** \\ \hline
1 & TbPO\({}_{4}\)H\({}_{2}\)O@silica-NH\({}_{2}\) -CD133 mAb & 40 \(\mathrm{\SIUnitSymbolMicro}\) & 0 & 100\% \\ \hline
2 & TbPO\({}_{4}\)H\({}_{2}\)O@silica-NH\({}_{2}\)-mAb’CD133-mAb’PD-L1 & 40 \(\mathrm{\SIUnitSymbolMicro}\) & 8–16 \(\mathrm{\SIUnitSymbolMicro}\) & 60–80\% \\ \hline \hline \end{tabular}
\end{table}
Table 1: The conjugated efficiency of TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\) nanorods with mAb against CD133 and PD-L1.  
Figure 5: NTERA-2 cells after 1 h of incubation with (**a**) TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\); (**b**) **TMC** (TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\)-mAb’CD133-mAb’PD-L1); (**c**) CD133-FITC; and (**d**) negative control; observed using an Olympus Scan’R 100X fluorescent microscope system.  
Figure 6: Flow cytometry analysis of labeled NTERA-2 cells incubated with (**a**) TbPO\({}_{4}\)\(\cdot\)H\({}_{2}\)O@silica-NH\({}_{2}\); (**b**) **TMC**, (**c**) CD133-FITC; and (**d**) negative control using a Novocyte flow cytometry system and NovoExpress software (ACEA Bioscience Inc.).  
noticed under the fluorescent microscopic observation. The results from flow cytometry analysis also showed a very low percentage of CD133-positive cells in the CCD-18 Co cell population (\(0.85\%\pm 0.07\%\)) which had been incubated with **TMC** (Figure 7). [PERSON] reported that the expression of CD133 in CCD-18 Co cells is hardly noticeable [12]. Thus, this cell line served as the negative control for CD133 markers. These results prove that the **TMC** nanocomplex could specifically bind to CSCs.