## 1 Introduction
The ship-in-a-bottle integration technique based on laser direct writing in a solution filled in a microchannel, even with complex shapes, can alleviate these restrictions. Here, we report multi-pattern and 3D integration of protein microstructures into Foturan(r) (Schott AG, Mainz, Germany) glass microchannels by femtosecond laser direct writing-induced cross-linking. An aqueous solution of protein mixed with photoinitiator is pipetted into a glass microchannel and a femtosecond laser is focused in the sample on a personal computer (PC)-controlled stage with in situ observation using a charge-coupled device (CCD) camera (Figure 1a). The cross-linking mechanism is schematically shown in Figure 1b. When the laser irradiates the solution containing protein and photoinitiator, photoinitiator molecules are excited at the laser focus by multi-photon absorption, initiating protein molecules to become radicals. For the 3D fabrication, the multi-photon absorption processes are essential [16]. The probability of multi-photon absorption processes is strongly dependent on the laser intensity. For instance, the absorption cross-section for n-photon absorption is proportional to the n-th power of the laser intensity. Therefore, the multi-photon absorption can be efficiently induced only at a laser intensity above a specific critical value, which is dependent on both the material and the pulse width. When a femtosecond laser beam is focused inside a transparent material with an adequate pulse energy, the multi-photon absorption can be confined to a region near the focal point inside the material where the laser intensity exceeds the critical value. In this way, the internal modification of transparent materials and the fabrication of structures inside them can be performed, which enables us to create 3D structures by scanning the focused femtosecond laser beam inside the transparent materials. We generate green laser light as a second harmonic from near infrared lasers, which is transparent the glass used as microfluidic device platforms, the protein, and the photoinitiator. Subsequently, a radical chain reaction creates more radicals amongst protein molecules. While most radicals are probably relaxed due to interaction with solvent molecules, some protein radicals cross-link intermolecularly to form the proteinaceous element along the PC-controlled path of light exposure. Radicalization and subsequent cross-linking are considered to be random within a protein [11]. While femtosecond laser polymerization has a reduced risk of protein denaturation due to little heat generation, the random cross-linking might disrupt functional domains or protein tertiary structure for a subset of the protein inside the fabricated microstructure. The impact of such a disruption might depend on the structural robustness of the respective protein. Cell adhesion [10; 11] and selective binding [11; 12; 15] have been reported to indicate that protein function was retained. In this study, we demonstrate the integration of BSA in the glass microfluidic channels. BSA is an abundant, stable, and soluble protein from blood plasma [17], which is routinely used in 3D microfabrication [8; 9; 10; 11; 12; 13; 14; 15]. As a photoinitiator, we utilize (Sodium 4-[2-(4-Morpholino) benzoyl-2-dimethylamino] butylbenzenesulfonate) (MBS) because it is a water-soluble variant of the widely used Irgacure 369 with a broad UV absorption peak [18; 19]. MBS is free of toxic heavy metals and requires no organic solvents [20]. We further demonstrate the micropatterning of multi-proteins by a two-step integration of enhanced green fluorescent protein (EGFP) and BSA. EGFP, which is an engineered variant of GFP, is widely used for bioimaging [21]. However, the fabrication resolution  
Figure 1: Schematic illustration of concept of proteinaceous microcomponent integration inside a closed microfluidic channel. (**a**) Illustration shows a three-dimensional (3D) glass microchannel with inlet and outlet, covered and filled with a drop of protein solution.