## 2 Materials and Methods
### Microfluidic System and Tests  
To extend the demonstration of the feasibility of this fluorescence-based biomarker detection in a microfluidic-sensing device, a microfluidic system was developed. This system provides a platform to study the Alzheimer's-related biomarker A\(\beta\) at a miniaturized scale, while preserving the optical and fluidic precision established in macroscale experiments. The microfluidic device was constructed using polydimethylsiloxane (PDMS) due to its optical transparency, biocompatibility, and scalability [28; 29]. PDMS's transparency across a wide range of wavelengths ensures minimal interference with fluorescence detection, while its ease of fabrication supports the creation of intricate microchannel designs. The fabricated device consists of a fluid chamber tailored for fluorescence observation, with an inlet and outlet for delivery and removal of reagents. Key design features included a circular well with a diameter of 8 mm and a height of 3 mm, coupled with inlet and outlet channels that are 2 mm wide, the same dimensions of the chamber used to culture tissue in previous OOC studies, as reported by [PERSON] et al. (2024) [28]. The microchannel system maintains a steady laminar flow and prevents turbulence that could interfere with fluorescence signal stability. Figure 2 illustrates the CAD design and 3D layout of the microfluidic system, highlighting the chamber's capacity to support consistent sample distribution for fluorescence detection. The microfluidic device was fabricated using a soft lithography technique, following the standard fabrication steps [30]. Finally, the microfluidic devices were closed with a glass slide by oxygen plasm for 30 s at 30 W (Plasma Systems ZEPTO from Diener electronic (Ebhausen, Germany)). This procedure, besides creating the permanent sealing of the microfluidic devices with the glass, allows for the hydrophilization of PDMS, avoiding possible unwanted binding of organic molecules to the surface microfluidic channels. For the fluorescence experiments, this device was integrated into a custom-designed setup created with Fusion AutoCAD software (version 2.0.20508). This setup was engineered to ensure precise component alignment and to minimize light interference during fluorescence detection. As illustrated in Figure 2, the incident light and the collected emitted fluorescence are aligned on the same line, due to the miniaturization constraints, e.g., avoiding the circular side of the microfluidic chamber, which would compromise the reproducibility of the measurements. Additionally, the system comprises a black enclosure (colored in gray for better visualization) with a removable lid and two internal levels. The upper level houses the microfluidic device, while the lower level holds a support for the optical filter. Furthermore, two optical fibers are carefully aligned and attached to collimating lenses for the illumination and fluorescence collection to/from the microfluidic device. Excitation light is directed into the well via an optical fiber inserted through the top of the box, while fluorescence emitted from the sample is collected by a second optical fiber attached at the bottom of the box. Each microfluidic chip is designed with two independent optical-reading wells, in which the excitation and detection fiber holes are strategically positioned to optimize fluorescence collection from one well at a time. Unlike the macroscale setup, the optical system in this configuration is aligned at a 180\({}^{\circ}\) angle, with the long-pass filter to effectively block the excitation light. This precise alignment ensures that only the emitted fluorescence is collected. Nonetheless, the same light source and detector (spectrophotometer and software) are used to obtain results from the present experiments. For the fluorescence detection, CRNAAD-2 and A\(\beta\) samples were pre-mixed (1/1, \(v/v\)) and introduced in the inlet of the microfluidic device using a syringe through the inlet.