# Single-Cell Patterning Based on Immunocapture and a Surface Modified Substrate
## 2 Preparation of the Substrate for Single Cell Patterning
### PEG-Silane Passivation Process  
The nonselective adhesion of the bovine serum protein in the following protein adhesion process renders the cleaning process difficult and the pattern quality degraded if the HMDS treated slides are not further treated. Here, we used poly(ethylene glycol)-silane (PEG-silane) as a passivator to treat the substrate with no siloxane groups, thus preventing it from bovine serum albumin adhesion. The contact angle test showed that the passivation treatment of PEG-silane did not affect the HMDS self-assembled molecular layer formed on the slide. Here, the contact angle was also measured using the OCA20 contact angle measurement instrument of DataPhysics, Fielderstadt, Germany. With the high hydrophilic polymer chain, PEG can significantly reduce the adhesion of hydrophobic proteins on the substrate. Its reaction mechanism is shown in Equation (2). PEG-silane passivation technology is extremely simple: It only requires the immersion of the slide in the solution containing PEG-silane for 2-4 h to complete PEG-silane grafting on the exposed glass surface, to form a relatively hydrophilic self-assembling molecular layer. \[ \begin{array}{c}6(\text{Si}_{(substrate)}-OH)+2[CH_{3}O-(\text{C}_{2}H_{4}O)_{6 -9}-(CH_{2})_{3}Si(OCH_{3})_{3}]\rightarrow\\ 2[(Si_{(substrate)}O)_{3}-Si(CH_{2})_{3}(\text{C}_{2}H_{4}O)_{6-9}-OCH_{3}]+6 CH_ {4}+3O_{2}\end{array}  \tag{2}\]  
The authors of [42] conducted a study on the concentration of the PEG-silane solution, and concluded that PEG-silane exhibited a better passivation effect at 10 mM concentration, which is in accordance with our experiment results. Here, we used the PEG-silane {2-[Methoxy (Polyethyleneoxy)Propyl]JTrimethoxySilane} from Gelest, and the molecular weight is 459-591. In this  
Figure 3: Influence of the heating time on the surface contact angle. By varying the heating temperature and heating time, by heating the substrate under 110 \({}^{\circ}\)C and 150 \({}^{\circ}\)C for 20 s, 40 s, 60 s, 80 s, 100 s, 150 s, 200 s, 300 s, 400 s, 500 s, and 600 s at each temperature, the deionized water contact angles were measured in different heating parameters. experiment, 1 mM to 10 mM of PEG-silane solution was added in the anhydrous toluene solution, and 1% of triethylamine catalyst solution was also added. Subsequently, the substrate was immersed in the solution at room temperature for 2 h. Subsequently, it was ultrasonically cleaned with toluene for 5 min, followed by rinsing with isopropyl alcohol and deionized water, and drying. The contact angle test showed that the substrate with a 1 mM concentration of PEG solution passivation has a smaller contact angle, which is 46\({}^{\circ}\)\(\pm\) 3\({}^{\circ}\), while the contact angle of the substrate treated with the 10 mM concentration of PEG is 42\({}^{\circ}\)\(\pm\) 3\({}^{\circ}\), as shown in Figure 4.  
Figure 5 shows the fluorescence micrograph after the adhesion of bovine serum protein and streptavidin. The left image is the result of passivation in the PEG-silane solution with the concentration of 1 mM, and the right figure is the passivation result of the 10 mM solution. The background of the left image shows nonselective adhesion, and the nonselective adhesion in the contrast pattern on the right is lower. We further investigated the result of PEG passivation. We found that the HMDS self-assembled molecular layers can enhance protein adhesion, while the substrate after PEG passivation inhibited protein adhesion well (Supplementary materials Figure S1). Finally, the passivation process was determined, in which the substrate was immersed in PEG-silane solution of concentration 10 mM for 2 h.  
The parameters optimization results of the substrate preparation process for single-cell patterning are shown in Table 1.  
Figure 4: Changes in surface contact angle before and after poly(ethylene glycol) (PEG) passivation. The contact angle without passivation is 68\({}^{\circ}\)\(\pm\) 3\({}^{\circ}\).