Nova plataforma microfluídica multiplexer para diagnóstico molecular rápido de acidente vascular cerebral

MOTIVATION: Current stroke assessment is mainly based on clinical evaluation and neuroimaging. Stroke standard management is divided in pre-hospital, in-hospital, post-hospital, and time delays within the stroke survival chain workflow are associated with worse outcomes (Fig.3). Also, conventional methods for blood biomarkers quantification are invasive, time and reagent consuming, involve multiple steps (reaction/washing), being inappropriate for portable devices. These limitations motivate the development of automatic miniaturized microfluidic platforms to simultaneously monitor various stroke biomarkers, where fast diagnosis is critical for the patients’ outcome. Thus, the potential use of PoC devices based on blood-derived biomarkers to aid clinical decision-making for the stroke diagnosis, patients triaging for acute reperfusion therapies, and in informing stroke mechanisms and prognosis, has shown to be a great addition to a faster assessment of clinical decision-making. OBJECTIVES: This project aims developing a PoC, portable microfluidic device, to shorten the time to treatment decision and improving patient’s outcomes by fulfilling an unmet need in stroke management. The major challenge addressed is to develop an original highly sensitive, multiplexer LOC for sample pre-processing, diagnosis and risk stratification of stroke patients, based on simultaneous quantification of 3 biomarkers in minimally invasive body fluids (GFAP, MMP-9, TnI will be targeted)(Fig.1). The self-operable microfluidic biosensor platform will integrate on-chip sample passive and acoustic-based pre-processing and control, multiplexer microfluidics, parallel competitive immunoassays, optical detection and control microelectronics for low cost, portable, simultaneous detection of 3 biomarkers, ensuring small sample/reagents volumes, automation, rapid processes. Competitive Ab-based assays allow specific targeting of GFAP, MMP-9 and TnI in the sensor polymeric surface, which will be detected by grating-enabled high efficiency photodiodes in CMOS technology. A decision algorithm will gather information from the biomarkers, relating them with the onset of stroke. The on-chip integrated system will bring huge impact and provide a considerable benefit to the healthcare, in emergency rooms and even remote regions that may lack sophisticated laboratory facilities and/or highly-trained personnel. SPECIFIC GOALS: -fabrication of multiplexer microfluidic die for whole blood (fingerprick) pre-processing, cells filtration and plasma driving into selected channels; -innovative piezoelectric PVDF patterning in microfluidic substrate for selective fluid pumping over the channel; -simultaneously highly specific biorecognition of GFAP, MMP-9 & TnI in the sensor surface with pg/ml detection range, competitive with ELISA; -patterning, fabrication, characterization of high quantum efficiency Si photodiodes (>40% at 450nm); -on-chip integration of optical detection and control with the microfluidic system for automatic PoC measurement in minimum blood volumes; -simple decision algorithm integrated for fast and sensitive stroke assessment; -potential to further detect more relevant biomarkers in the same LOC and detection system, only replacing the disposable PDMS die by others with alternative selections of immobilized antibodies in the die substrate (NFL, TNF-a, CRP, BNP,etc); -Validation of the microfluidic biosensing system for fast, minimally invasive stroke diagnosis.