## 3 Results and Discussion
### Microfluidic-Sensing Studies for Amyloid-Beta Analysis
#### 3.2.3 Optimizing Binding Studies of CRANAD-2 with Amyloid-Beta in a Microfluidic Device  
One of the goals of this study was to explore the viability of the microfluidic platform for detecting A\(\beta\) pushing the boundaries of previous detection ranges and approximating the variability reported in the literature for A\(\beta\) aggregate variations in the presence of AD [35; 36]. To achieve this, the experimental setup was optimized by reducing the concentrations to ranges between 5 nM and 100 nM. Figure 4e,f illustrate the results obtained at these lower concentrations, revealing a consistent trend. As in the previous experiments, oligomeric A\(\beta\) exhibits an inverse relationship between concentration and fluorescence intensity, whereas fibrillar A\(\beta\) shows a positive correlation. The results demonstrate that even with an optical path miniaturized, the microfluidic-sensing system presented in this work is able to effectively differentiate a lower concentration range of A\(\beta\) (5 nM). While absolute fluorescence signals were attenuated in the microscale environment, fibrillar A\(\beta\) consistently yielded higher signals compared to oligomers across all tested concentrations. These results serve as a strong validation for the microfluidic system's potential, providing a foundation for further optimization and exploration of its capabilities at low concentration levels, which are more in line with the ones found in vivo [34; 35; 36]. To fully understand the luminescence difference at wavelength 736 nm between the different forms of the protein, a bar graph for normalized relative difference is presented in Figure 5a. Here, it can be seen that even for the highest light intensity scenarios in both forms, oligomers did not yield even 50% of the light emitted by fibrils. Additionally, since the fibrilated A\(\beta\) structures present as the pathological hallmark of [PERSON]'s disease, this specificity supports the use CRANAD-2 as an optical reporter to detect and quantify fibrilated A\(\beta\) with a high potential for in vitro AD studies. Moreover, the normalized fluorescence intensities, shown in Figure 5b, further emphasize the consistent results between oligomeric and fibrillar A\(\beta\). Similar to the macroscale results, the relative difference in fluorescence intensity is predominantly attributed to the fluorescence produced by A\(\beta\) fibrils. These findings support the successful transition of CRANAD-2 from macroscale to microfluidic platforms for fluorescence-based A\(\beta\) detection. While absolute fluorescence intensities were lower in the microfluidic system and signals were more affected by noise, the probe's reliable fluorescence response across concentrations confirms its potential for use in advanced microfluidic biosensing applications. This demonstrates that, despite the challenges posed by the microfluidic environment, CRANAD-2 retains its sensitivity and specificity, making it a viable candidate for scalable, low-volume biomarker detection in Alzheimer's disease diagnostics. Figure 5c examines the normalized intensity difference for a broader range of lower A\(\beta\) concentrations in the microfluidic setup. Unlike the pronounced disparity observed in panels (a) and (b), the differences between fibrillar and oligomeric A\(\beta\) become less distinct in this range. However, the clear trends of increasing fluorescence for fibrillar A\(\beta\) and decreasing fluorescence for oligomeric A\(\beta\) across the tested concentrations are  
of particular interest. These trends validate the probe's capability to detect even subtle changes in fluorescence, which is crucial for precise biomarker quantification in early detection scenarios. Thus, while the magnitude of the difference is less pronounced, this panel underscores the robustness of CRANAD-2 in capturing concentration-dependent fluorescence variations, further confirming its suitability for scalable, low-concentration biomarker detection in microfluidic platforms.