## 3 Results and Discussion
### Fabrication of Myriad of Microchambers in PDMS by Using PG Molds  
Studies showed that when compared with conventional methods, the use of microwell biochips in bio-nanotechnologies could result in 100- to 1000-fold reduction in the number of cells required for analyses due to great reduction of microchamber volume, down to \(<\)1 nL [7]. We advance herein the possibility to fabricate up to 10,000 microchambers in PDMS in a single step based on a PG mold processed by PLAE. The protocol is schematically depicted in Figure 4. First, we applied PLAE in order to create 3D pillars in PG after the chemical etching of laser-exposed regions. In particular, by scanning transversal lines onto PG surface, one can design a chess-like board with square dimensions tailored by the CAD (computer-aided design) program by a maskless process (Figure 5a,b). The laser-irradiated regions were designed so as to develop desired depth dimensions. Specifically, one can irradiate either with a multilayer configuration if deep channels are required or with a single-layer configuration for superficial structuring. Similarly to Section 3.1, the laser irradiation process is followed by annealing treatment at 605 \({}^{\circ}\)C and chemical etching in HF solution. Figure 5c,d shows the exposed PG after annealing treatment where the brown color represents  
Figure 3: Microfluidic chip fabricated by PLAE in Foturan glass. Photographic images of the entire structure (**a**) after laser scanning and annealing, (**b**) after chemical etching in HF, and (**c**) developed chip (1.5 \(\times\) 1.5 cm\({}^{2}\)) connected to perfusion system that can switch between three different reagents and provides a continuous flow; (**d–f**) detailed magnified images from the regions highlighted in dotted squares in (**a–c**), respectively. modified PG and the white areas are the unmodified square structures. During the chemical etching the modified PG was selectively removed so that pillars could be created from the unmodified square regions whose heights can be controlled with the etching time. The etching rate was constant regardless of times and depths since the sufficiently deep structures were modified and the applied laser dose was kept constant within the modified structures. We, thus, obtained PG molds with 2500 pillars of \(150\times 150\times 60\) um\({}^{3}\) or even with 10,000 pillars of \(50\times 50\times 30\) um\({}^{3}\) (length \(\times\) width \(\times\) height). We used the fabricated PG molds to obtain replicas in PDMS, by which the pillar-prints were transcribed, i.e., microchambers with the same X-Y dimensions of glass pillars and depth corresponding to the glass pillar height. All processes are schematically shown in Figure 4.  
Sketches and photographs of PG after the laser irradiation and the annealing treatment are shown in Figure 5a,b, while optical images of the exposed patterns (single-layer laser scanning) are visualized in Figure 5c,d. Similarly, Figure 5e,f depicts the mold sketch and photograph after chemical etching and Figure 5g,h presents magnified images of Figure 5f. Figure 4: Schematic of the fabrication procedure of the PG (photosensitive glass) mold and of 3D microchambers in PDMS. The mold was obtained after the PG processing protocol by PLAE, described in Section 2. The PDMS replica was obtained after 24 h drying at room temperature upon separating the PDMS (Polydimethylsiloxane) from the mold. Figure 5: Fabrication of PG glass mold. PG after laser exposure and annealing treatment: (**a**) sketch of the irradiated patterns; (**b**) photograph of the entire sample; (**c**) optical image of central region; (**d**) detailed view of (**c**). PG after chemical etching; (**e**) 3D mold sketch; (**f**) photograph of the entire PG mold; (**g**) optical image of central region; (**h**) detailed view of (**g**). Using the fabricated mold, one can get controlled and efficient synthesis of thousands of microchambers having a dimension of \(150\times 150\times 60\)\(\upmu\)m\({}^{3}\) (Figure 6a).