*3.3. Integration of High-Resolution Microstructures*

For a broader application scope, we investigated the potential to incorporate microstructures within 3D printed channels. In order to do so, a process was developed to allow for the integration of microstructures into the FDM printed microfluidic structures (see Figure 4a). To generate the desired microstructures, we used a photocurable PMMA prepolymer, which was structured on top of a commercial PMMA substrate. This PMMA substrate was then used as the PMMA substrate plate for FDM printing. This requires careful alignment of the PMMA substrate with the PMMA microstructure in the FDM printer to allow a precise print-on process. We first replicated a microscale lines-and-space structure using the PMMA prepolymer (Figure 4b), and then printed a channel around it, resulting in a channel with integrated microstructures (see Figure 4c). By doing so, we achieved the integration of high-resolution PMMA microstructures within the FDM printed PMMA channels.

*Micromachines* **2020**, *11*, 873 8 of 13

**Figure 3.** Microfluidic chips printed in PMMA. (**a**) Microfluidic mixer cascade for effective mixing of dyed water (scale bar: 10 mm). (**b**) 2.5-dimensional microfluidic spiral (scale bar: 10 mm). (**c**) Enhanced mixer structure of 600 µm periodic structures, effectively mixing dyed water (scale bar: 10 mm). (**d**) Three-dimensional microchannel printed in PMMA around a straight channel, visualized using an aqueous fluorescent dye (scale bar: 10 mm). **Figure 3.** Microfluidic chips printed in PMMA. (**a**) Microfluidic mixer cascade for effective mixing of dyed water (scale bar: 10 mm). (**b**) 2.5-dimensional microfluidic spiral (scale bar: 10 mm). (**c**) Enhanced mixer structure of 600 µm periodic structures, effectively mixing dyed water (scale bar: 10 mm). (**d**) Three-dimensional microchannel printed in PMMA around a straight channel, visualized using an *Micromachines* aqueous fluorescent dye (scale bar: 10 mm). **2020**, *11*, 873 9 of 13

channels. **Figure 4.** Integration of high-resolution PMMA structures inside a FDM-printed channel structure. (**a**) Principle of integration of high-resolution microstructures inside the FDM printed channel. The microstructure is fabricated by polymerizing a PMMA prepolymer directly on a commercial PMMA substrate using a lines-and-space structure. The PMMA substrate is then aligned and fixed within the FDM printer and the microfluidic channel is printed around the microstructure. (**b**) Polymerized PMMA microstructure fabricated using photocurable PMMA prepolymers (scale bar: 100 µm). (**c**) FDM printed PMMA chip including the microstructure from (**b**) (scale bar: 10 mm). The inset shows the integrated microstructure (scale bar: 100 µm). **Figure 4.** Integration of high-resolution PMMA structures inside a FDM-printed channel structure. (**a**) Principle of integration of high-resolution microstructures inside the FDM printed channel. The microstructure is fabricated by polymerizing a PMMA prepolymer directly on a commercial PMMA substrate using a lines-and-space structure. The PMMA substrate is then aligned and fixed within the FDM printer and the microfluidic channel is printed around the microstructure. (**b**) Polymerized PMMA microstructure fabricated using photocurable PMMA prepolymers (scale bar: 100 µm). (**c**) FDM printed PMMA chip including the microstructure from (**b**) (scale bar: 10 mm). The inset shows the integrated microstructure (scale bar: 100 µm).

reported to be between 65° and 72° [32] and underline the hydrophilic character of the material.

The wetting behavior of the chip material is important in microfluidics, to enable, e.g., filling of the channels and thus capillary-driven microfluidics in general. Therefore, the contact angle of water

The optical transmission of the 3D printed PMMA with the different nozzle distances described in Section 3.1, was characterized using UV/Vis measurements (see Figure 5a). Samples with a thickness of 100 µm, 400 µm and 1 mm were measured. The transparency was compared to the optical transmission of commercial PMMA with a thickness of 2 mm, which was used as the substrate for 3D printing of microfluidic channels directly on top of the substrate. The optical transparency of FDM printed PMMA parts was increased by using a printing strategy with reduced nozzle distance compared to the standard distance. The optical transmission, however, is still below that of commercial PMMA substrates. This is why for applications where high optical transmission is required in the region of interest, the demonstrated strategy of printing on top of a commercial

To ensure the applicability of 3D printed PMMA for fluorescence-based assays, the influence of autofluorescence of the material was investigated using an inverted fluorescence microscope. The printed and the commercial substrate show comparable low autofluorescence values (see Figure 5b).

*3.4. Characterization of Printed PMMA* 

PMMA substrate is preferred.

### *3.4. Characterization of Printed PMMA* the channels and thus capillary-driven microfluidics in general. Therefore, the contact angle of water was measured on a commercial PMMA substrate and on a 3D printed substrate to characterize the

the integrated microstructure (scale bar: 100 µm).

The wetting behavior of the chip material is important in microfluidics, to enable, e.g., filling of the channels and thus capillary-driven microfluidics in general. Therefore, the contact angle of water was measured on a commercial PMMA substrate and on a 3D printed substrate to characterize the wetting behavior. The printed PMMA and the commercial PMMA showed a water contact angle of 69◦ , respectively. These values are in good accordance with literature references which have been reported to be between 65◦ and 72◦ [32] and underline the hydrophilic character of the material. wetting behavior. The printed PMMA and the commercial PMMA showed a water contact angle of 69°, respectively. These values are in good accordance with literature references which have been reported to be between 65° and 72° [32] and underline the hydrophilic character of the material. The optical transmission of the 3D printed PMMA with the different nozzle distances described in Section 3.1, was characterized using UV/Vis measurements (see Figure 5a). Samples with a thickness of 100 µm, 400 µm and 1 mm were measured. The transparency was compared to the optical

The wetting behavior of the chip material is important in microfluidics, to enable, e.g., filling of

*Micromachines* **2020**, *11*, 873 9 of 13

**Figure 4.** Integration of high-resolution PMMA structures inside a FDM-printed channel structure. (**a**) Principle of integration of high-resolution microstructures inside the FDM printed channel. The microstructure is fabricated by polymerizing a PMMA prepolymer directly on a commercial PMMA substrate using a lines-and-space structure. The PMMA substrate is then aligned and fixed within the FDM printer and the microfluidic channel is printed around the microstructure. (**b**) Polymerized PMMA microstructure fabricated using photocurable PMMA prepolymers (scale bar: 100 µm). (**c**) FDM printed PMMA chip including the microstructure from (**b**) (scale bar: 10 mm). The inset shows

The optical transmission of the 3D printed PMMA with the different nozzle distances described in Section 3.1, was characterized using UV/Vis measurements (see Figure 5a). Samples with a thickness of 100 µm, 400 µm and 1 mm were measured. The transparency was compared to the optical transmission of commercial PMMA with a thickness of 2 mm, which was used as the substrate for 3D printing of microfluidic channels directly on top of the substrate. The optical transparency of FDM printed PMMA parts was increased by using a printing strategy with reduced nozzle distance compared to the standard distance. The optical transmission, however, is still below that of commercial PMMA substrates. This is why for applications where high optical transmission is required in the region of interest, the demonstrated strategy of printing on top of a commercial PMMA substrate is preferred. transmission of commercial PMMA with a thickness of 2 mm, which was used as the substrate for 3D printing of microfluidic channels directly on top of the substrate. The optical transparency of FDM printed PMMA parts was increased by using a printing strategy with reduced nozzle distance compared to the standard distance. The optical transmission, however, is still below that of commercial PMMA substrates. This is why for applications where high optical transmission is required in the region of interest, the demonstrated strategy of printing on top of a commercial PMMA substrate is preferred. To ensure the applicability of 3D printed PMMA for fluorescence-based assays, the influence of autofluorescence of the material was investigated using an inverted fluorescence microscope. The printed and the commercial substrate show comparable low autofluorescence values (see Figure 5b).

**Figure 5.** Characterization of FDM printed PMMA. (**a**) UV-Vis measurement of printed PMMA using different printing strategies (standard and reduced nozzle distance) and commercial PMMA substrates. (**b**) Fluorescence intensity of commercial PMMA substrate and FDM printed PMMA.

To ensure the applicability of 3D printed PMMA for fluorescence-based assays, the influence of autofluorescence of the material was investigated using an inverted fluorescence microscope. The printed and the commercial substrate show comparable low autofluorescence values (see Figure 5b).

### *3.5. Biofunctionalization Inside the Printed Microfluidic PMMA Chips*

Protein patterns with specific shapes and patterns are capable of inducing specific cellular responses and are important for the study of cellular behavior. To demonstrate the possibility to locally create these patterns within the printed PMMA channels, we used a fast protein patterning method based on the immobilization of fluorescently labeled molecules via photobleaching which we have previously reported [28]. The photochemical immobilization of fluorescently labelled biomolecules is based on the transformation of fluorophores into reactive radicals as a consequence of photobleaching upon irradiation at the absorption wavelength. The radical species, which are only formed in the exposed areas, react with functional groups and thus bond covalently to the surface. The method is shown in Figure 6a. In this work, we created the pattern directly within the channel by printing a microfluidic PMMA channel on top of a transparent PMMA substrate (shown in Figure 6b), filling it with fluorescently labelled biotin (F5B) and illuminating it with a maskless projection lithography system. The patterns of photobleached F5B were visualized by staining with STV-Cy3. Biomolecule patterns with feature sizes of 125 µm were fabricated inside the microfluidic channel showing the facile biofunctionalization of 3D printed PMMA microfluidics (Figure 6c,d).

showing the facile biofunctionalization of 3D printed PMMA microfluidics (Figure 6c,d).

**Figure 5.** Characterization of FDM printed PMMA. (**a**) UV-Vis measurement of printed PMMA using different printing strategies (standard and reduced nozzle distance) and commercial PMMA substrates. (**b**) Fluorescence intensity of commercial PMMA substrate and FDM printed PMMA.

Protein patterns with specific shapes and patterns are capable of inducing specific cellular responses and are important for the study of cellular behavior. To demonstrate the possibility to locally create these patterns within the printed PMMA channels, we used a fast protein patterning method based on the immobilization of fluorescently labeled molecules via photobleaching which we have previously reported [28]. The photochemical immobilization of fluorescently labelled biomolecules is based on the transformation of fluorophores into reactive radicals as a consequence of photobleaching upon irradiation at the absorption wavelength. The radical species, which are only formed in the exposed areas, react with functional groups and thus bond covalently to the surface. The method is shown in Figure 6a. In this work, we created the pattern directly within the channel by printing a microfluidic PMMA channel on top of a transparent PMMA substrate (shown in Figure 6b), filling it with fluorescently labelled biotin (F5B) and illuminating it with a maskless projection lithography system. The patterns of photobleached F5B were visualized by staining with STV-Cy3.

*3.5. Biofunctionalization Inside the Printed Microfluidic PMMA Chips* 

**Figure 6.** Biofunctionalization inside the 3D printed PMMA channel. (**a**) Fluorescently labelled biomolecules can be covalently bound to surfaces by photobleaching the fluorophore upon exposure with light patterns. (**b**) 2 mm wide embedded PMMA channel printed on a PMMA substrate with biofunctionalization inside the printed channel indicated by dotted lines (scale bar: 10 mm). (**c**) The bottom surface of the 3D printed channel on top of the commercial PMMA slide was functionalized with different biotin patterns by photobleaching of F5B. Patterns were illuminated for 3 min (patterns 1 and 2) and 5 min (patterns 3 and 4). The patterns were visualized via staining with STV-Cy3 (scale bar: 750 µm). Signal-to-noise ratios were calculated for all four images and were on average 29 for 3 min illumination and 11 for 5 min. (**d**) Close-up of patterns which were illuminated for 3 min. **Figure 6.** Biofunctionalization inside the 3D printed PMMA channel. (**a**) Fluorescently labelled biomolecules can be covalently bound to surfaces by photobleaching the fluorophore upon exposure with light patterns. (**b**) 2 mm wide embedded PMMA channel printed on a PMMA substrate with biofunctionalization inside the printed channel indicated by dotted lines (scale bar: 10 mm). (**c**) The bottom surface of the 3D printed channel on top of the commercial PMMA slide was functionalized with different biotin patterns by photobleaching of F5B. Patterns were illuminated for 3 min (patterns 1 and 2) and 5 min (patterns 3 and 4). The patterns were visualized via staining with STV-Cy3 (scale bar: 750 µm). Signal-to-noise ratios were calculated for all four images and were on average 29 for 3 min illumination and 11 for 5 min. (**d**) Close-up of patterns which were illuminated for 3 min.

### **4. Conclusions**

In this work, we demonstrated that microfluidic PMMA chips can be fabricated down to channel diameters of 300 µm using a benchtop FDM machine. It was shown that the transparency of the printed chips can be significantly improved by using a commercial PMMA substrate as the bottom substrate for the printing process. The 3D printed PMMA was compared to commercial PMMA and shows the same low autofluorescence and hydrophilic wetting behavior, which is important for biocompatibility and channel wetting. We demonstrated that the integration of microstructures, as well as biofunctionalization in 3D printed PMMA channels, is possible. We showed that high-resolution microstructures can be produced using a simple prepolymer-casting process and that these structures can be easily integrated into the channel by directly printing onto the structured PMMA substrate. We have also shown that patterns of biomolecules can be attached to the commercial PMMA substrate using a photobleaching process. This effectively enables the formation of microfluidic channels with high-resolution biofunctionalization within the channel itself.

This work will allow the use of PMMA, one of the most important and industrially-relevant thermoplastic polymer materials in microfluidic mass-market manufacturing, in early stages of component development, where rapid prototyping and 3D printing facilitate fast design iterations and rapid experimental verifications. This closes an important gap in the academia-to-industry transition by allowing usage of the same material both during early-stage lab-scale development and industrial-scale mass-market production.

**Author Contributions:** F.K. concept idea, design of experiments and writing of the paper, N.D. and M.M. printing experiments, A.K. Biofunctionalization, D.H., M.M., P.R. and B.E.R. UV-Vis measurements and contribution to the writing of the paper. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was partly funded by the German Ministry of Education and Research (BMBF), funding code 03×5527 "Fluoropor". This research was partly funded by the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG) for funding through the Centre for Excellence livMatS Exec 2193/1-390951807. This work was partly funded by the Research Cluster "Interactive and Programmable Materials (IPROM)" funded by the Carl Zeiss Foundation. This work was partly financed by the Baden-Württemberg Stiftung gGmbH within the program "Biofunctional Materials and Surfaces" (BiofMo-2, 3D Mosaic).

**Conflicts of Interest:** The authors declare no conflict of interest.

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