A-395 From Blot to Byte: Traumatic Brain Injury (TBI) Diagnosis and Monitoring using a Portable, Precise, and Ultrasensitive, Digitalized Western Blot (dWB)

Abstract Background Traumatic brain injury (TBI) remains a leading cause of disability and death in adults, underscoring the need for reliable diagnostic methods. Quantitative detection of biomarkers such as glial fibrillary acidic protein (GFAP) and ubiquitin C-terminal hydrolase-L1 (UCH-L1) is critical for accurately diagnosing and monitoring TBI. Although traditional Western blotting offers high specificity for protein analysis, its complicated settings and inability to precisely quantify low-abundance targets limit its clinical utility—particularly in monitoring changes of biomarker levels at point-of-care (POC) settings. To address these challenges, we developed an integrated digitized Western blot (dWB) microfluidic platform that combines the accessibility of conventional WB reagents with the enhanced precision and sensitivity of digital assays. By integrating this approach into a microfluidic system and employing deep learning algorithms for data analysis, our platform promises rapid and accurate biomarker quantification tailored for clinical applications. Methods Microarray chips featuring an array of approximately 100×100 microwells (20µm in diameter with 20µm spacing) were fabricated as the foundation of the platform. Magnetic microbeads, functionalized with capture antibodies, were deployed to selectively isolate GFAP and UCH-L1 directly on the chip surface. Following target capture, the biomarkers were labeled using horseradish peroxidase-conjugated nanoparticles (HRP-NP). Upon introduction of an ultrasensitive TMB substrate, the HRP-NP catalyzed its oxidation, resulting in a localized colorimetric precipitate within the positive wells. Wells exhibiting pronounced color deposition were scored as “1” (signal), while those without observable change were assigned a “0”. Finally, a deep learning-based algorithm was applied to rapidly and accurately analyze the digital readouts. Results The dWB platform yielded distinct binary outputs, with target-positive wells displaying pronounced dark spots and minimal background interference. The HRP-NP effectively catalyzed the TMB reaction, resulting in concentrated color deposition precisely at the sites of target binding. This clear signal differentiation, marked by an elevated signal-to-noise ratio, highlights the platform*s capability for rapid and accurate biomarker quantification. Conclusion Our dWB platform harnesses conventional Western blotting reagents to achieve highly sensitive and precise protein biomarker detection. By delivering a clear binary output and integrating deep learning for rapid data analysis, this assay reliably identifies GFAP and UCH-L1, positioning the technology as a promising candidate for fully automated point-of-care diagnostics in TBI management.