Microsampling and point of care devices in biomarkers monitoring

Cardiovascular diseases (CVDs) refer to a group of conditions that affect the heart or blood vessels. According to the Centers for Disease Control and Prevention, CVDs represent the leading cause of death in the United States, resulting in 20.6% of total deaths. In the traditional model of diagnostic testing, the patient has to go to the hospital for sample collection, which is followed by sample processing, performing the test and then the doctor has to analyze the results and decide if the patient is to be admitted or discharged. By using Point of Care devices (POCs), everyone can perform a test at their own house, just by loading the sample and waiting for the results. POC is convenient for patients and clinicians, in terms of cost and time. The aim of this project was to develop a multiplex Luminex assay targeting neuronal and cardiovascular biomarkers and translate it into a portable device. For our study, we decided to focus our attention on cardiac and neuronal biomarkers such as: Human proBDNF, a member of the neurotrophic family, important since the heart is innervated by a sympathetic and parasympathetic system; proBDNF’s deficiency can lead to CVDs like high blood pressure, arrythmias, and atherogenesis, Human Galectin3, important for promoting ventricular remodeling and inflammation; GDF-15 (Growth differentiation factor-15) which has increased levels during cardiac inflammation and injury, and Human NTproBNP, N-terminal prohormone of BNP secreted in response to increased ventricular stretching. The first part of this study was performed by designing an assay for Luminex FlexMap 3D for biomarker detection, and selection of the antibody pairs. For each target, the first step was the creation of a suspension bead assay, in order to perform a singleplex and a multiplex analysis. LOD, LLOQ, ULOQ and CV values were calculated by using R software for both assays. In the second part of the study, we focus our attention on assay translation from Luminex FlexMap3D into microfluidic platforms. The first microfluidic device used was the PDMS chip, where some optimization steps were required in order to identify the type of beads and buffer to use, the amount of capture and detection antibody, and which method of signal generation to use. For the PDMS chip NHS-activated agarose beads were used with PBS buffer. The amount of capture antibody was determined by using a Rabbit IgG, in order to find the optimal concentration for the assay (31.25 ug). The concentration of the detection antibody was determinate by testing two different concentrations of each standard protein, in order to find the optimal condition for the assay. A colorimetric method of detection was used, in line with the idea of a portable device which allows to have rapid identification of the results. After the determination of the optimal conditions for the PDMS chip, a singleplex assay was performed; for each target the test was performed three times, and the LOD values were calculated. The second microfluidic device used was the Centrifugal Microfluidic Disc, and the assay was performed with the same conditions as the PDMS chip. Singleplex and multiplex assay was performed for each target, repeating each test three times for each target and LOD values were calculated. At the end of the study, the data obtained with the Luminex FlexMap 3D instrument and with the two different microfluidics devices was analyzed in order to understand how to improve the sensitivity/precision of the microfluidic devices to obtain a portable device, with promising results for Human NT-proBNP which showed LOD values similar to the one obtained with the Luminex Analysis.