Site-Selective Biofunctionalization of 3D Microstructures Via Direct Ink Writing

Abstract

Two-photon lithography has revolutionized multi-photon 3D laser printing, enabling precise fabrication of micro- and nanoscale structures. Despite many advancements, challenges still persist, particularly in biofunctionalization of 3D microstructures. This study introduces a novel approach combining two-photon lithography with scanning probe lithography for post-functionalization of 3D microstructures overcoming limitations in achieving spatially controlled biomolecule distribution. The method utilizes a diverse range of biomolecule inks, including phospholipids, and two different proteins, introducing high spatial resolution and distinct functionalization on separate areas of the same microstructure. The surfaces of 3D microstructures are treated using bovine serum albumin and/or 3-(Glycidyloxypropyl)trimethoxysilane (GPTMS) to enhance ink retention. The study further demonstrates different strategies to create binding sites for cells by integrating different biomolecules, showcasing the potential for customized 3D cell microenvironments. Specific cell adhesion onto functionalized 3D microscaffolds is demonstrated, which paves the way for diverse applications in tissue engineering, biointerfacing with electronic devices and biomimetic modeling.

Keywords: 3D cell culture; dip‐pen nanolithography; direct laser writing; phospholipids; proteins; surface functionalization; two‐photon lithography.

Figure 1

Schematic illustration of a) Two‐photon lithography process for 3D writing of microscaffolds using PETA and TPETA as monomers in the photoresist formulation and b) their biofunctionalization through DPN and/or µCS. c) SEM images of different microscaffolds for biofunctionalization, together with corresponding fluorescent images of the labelled phospholipid ink showing site‐specific functionalization.

Figure 2

a) Schematic representation of the surface modification of 3D microscaffolds with BSA and GPTMS, followed by direct ink writing through DPN. b) AFM images of printed phospholipid lines on 3D microscaffolds (PETA) with different surface modifications, right after printing and 1 week after. c) 3D AFM image of printed phospholipid dots on GPTMS treated PETA. Graph illustrating the relative volume loss of phospholipid dots printed on PETA and TPETA photoresists with different surface modifications due to ink spreading over 5 days. Scale bar 5 µm.

Figure 3

a) Schematic representations of i) biotinylated lipid printing on square structures via DPN followed by incubation with fluorescently labeled streptavidin, ii) AB printing via µCS, followed by incubation with fluorescently labeled secondary AB, and iii) µCS printing of fluorescently labeled fibronectin and as binding sites for cells. b) Optical microscopy images of structures with i) printed biotin bearing lipids as printed and after incubation with fluorescently labelled streptavidin, ii) printed antibodies before and after incubation with fluorescently labelled secondary antibodies, and iii) printed fluorescently labelled fibronectin before and after incubation with fibroblasts, labelled with DAPI.

Figure 4

a) Balb/3T3 fibroblasts on PETA square structures, either functionalized with fibronectin printed via µCS, or non‐functionalized (ctrl). Fluorescence imaging of the dotted square area shows the FN pattern, as well as clear positioning of the cells on the functionalized structures. b) Area coverage by cells in the case of functionalization and non‐functionalization, respectively (N = 3 samples, n = 120 structures per condition). c) SEM imaging of PETA tilted structures and corresponding confocal immunofluorescence imaging (maximum intensity projection over 12 µm). The cross‐section corresponding to the dotted line confirms cell adhesion on tilted surfaces in correspondence to the functionalized areas. Scalebar 50 µm.