# Single-Cell Patterning Based on Immunocapture and a Surface Modified Substrate
## 2 Preparation of the Substrate for Single Cell Patterning
### Surface Cleaning and HMDS Coating  
The physical and chemical properties of the substrate materials exhibit various influences on the lithography process. Particles that are not removed on the surface owing to insufficient cleaning will lead to tails or pinholes in the photoresist pattern, thereby affecting the adhesion force between the photoresist and the substrate; therefore, pattern defects, such as floating photoresist, will be generated after the development. In addition, the chemical properties of the substrate surface directly affect the adhesion of the photoresist and the substrate, resulting in the decrease in the quality and accuracy of the pattern. In our experiment, we found that the ultrasonic cleaning method cannot effectively remove the residues generated during the process, thereby resulting in the comet tail defect during the spinning process. Therefore, we used a piranha lotion (98% H\({}_{2}\)SO\({}_{4}\) and 30% H\({}_{2}\)O\({}_{2}\) mixed at a volume ratio of 7:3) in the ultrasonic cleaning for 10 min. Subsequently, the glass slide was rinsed with deionized water to remove the residues and blow dried using compressed air. The piranha lotion has a strong oxidation ability that can oxidize metal contaminants on the surface of the glass slide, thereby rendering them soluble in the solution. Meanwhile, the dehydration property of the sulfuric acid can carbonize organic substances, decomposing them into CO\({}_{2}\) and H\({}_{2}\)O with the strong oxidizing abilities of hydrogen peroxide. HMDS is a toxic, volatile, and colorless liquid that is harmful to human bodies. CVD is more suitable in this study, as the operator can be less exposed to HMDS. The primary concern is whether  
Figure 1: Surfacemodification process of the substrate. The photoresist mask was formed by direct lithography and the chemical vapor deposition (CVD) process was employed, followed by photoresist release and passivation treatment. HMDS: hexamethyldisilazane; PEG: polyethylene glycol. HMDS can penetrate the pores and react with the coated photoresist, and whether the additional heating process for photoresist drying will lead to difficulties in the photoresist release. By measuring the contact angle of the deionized water on the surface of the slide, the hydrophobicity of the material surface can be characterized, and the coating properties of the substrate can be evaluated. The contact angle was measured using the OCA20 contact angle measurement instrument of DataPhysics, Fielderstadt, Germany. After dry cleaning, the contact angle of the glass slide was 18 \(\pm\) 2\({}^{\circ}\). After HMDS vapor deposition, the contact angle increased to 75 \(\pm\) 4\({}^{\circ}\), demonstrating strong hydrophobic properties. The principle and the test results of HMDS vapor deposition are shown in Figure 2.  
In the early experiments, we used the typical parameters of HMDS coating in microfabrications technology as the CVD parameters. In this setting, the slide was baked on a baker for 15 min at 150 \({}^{\circ}\)C; subsequently, it was quickly placed into the liquid containing a small amount (approximately 1 mL) of HMDS in an airtight flask for CVD. After 15 min of cooling, the CVD process was completed, and the substrate was removed for the following photoresist release process. With such parameters, owing to the high temperature, the fluidity of the photoresist is strong, thus leading to the blockage phenomenon in the holes with diameters from 9 \(\upmu\)m to 10 \(\upmu\)m.Further, owing to the thermal denaturation of the photoresist, the releasing process could not be fully accomplished, and the residual photoresist inhibited the grafting of the passivation agent, thus resulting in nonselective adhesion. In view of these patterning defects, in the HMDS CVD process, the temperature and the time of heat treatment must be reduced while effectively depositing a thin HMDS layer. The substrate surface contact angle is related to the substrate cooling time in the HMDS steam.