Thesis presented October 05, 2023

Abstract:
Cardiovascular diseases are the leading cause of death in the world and several biomarkers are clinically used to diagnose patients. Cardiac troponin is currently the gold standard for the diagnostic of acute myocardial infarction (AMI). In hospital emergency departments, testing patient blood for cardiac troponin is crucial to rule-in or rule-out patients. Conventional methods to detect cardiac troponin and other biomarkers are usually performed in central medical laboratories with costly equipment and long analysis time. Point-of-Care devices propose on-site and rapid testing, but these devices currently lack sensitivity. We propose an innovative quantification assay for cardiac biomarkers integrated into a portable and automated instrument for rapid and high sensitivity testing.
We developed a method based on isothermal nucleic acids amplification (LAMP) using an original structure called dumbbell. For the quantification of biomarkers, the sample is incubated with this dumbbell structure containing various types of molecular probes such as aptamers or antibodies. Complete detection protocols are derived for various types of targets and integrated on a portable and automated platform. This latter contains microfluidic cartridges including a network of fluidic chambers used for the discrete manipulation of fluids. The cartridge architecture is designed specifically for the developed assay based on dumbbell amplification, and allow carrying out all the assay steps, from the sample recovery to the result.
We validated the isothermal amplification of the dumbbell structure with various types of molecular probes. We studied different parameters influencing the amplification reaction for a better understanding of the amplification process. We used thrombin as a model protein and included a specific thrombin aptamer in the dumbbell structure to obtain sensitive (100 pM) and antibody-free detection using this aptameroLAMP method. It is however limited to targets for which a double aptamer sandwich is available. For the troponin cardiac biomarker, we derived the method with antibodies coupled with the dumbbell structure for its exponential amplification, named immunoLAMP. We tested this method on human plasma samples and reached a limit of detection of 100 pg/mL, a relevant concentration for physio-pathological testing. Then, each step of the detection assay was successfully integrated in microfluidic cartridges on a portable instrument. Two main architectures were designed. The first one allowed the sample preparation on magnetic beads to sandwich capture the target. Troponin was detected down to 1 ng/mL in this cartridge using the fully automated instrument. The second architecture proposes the LAMP reaction integration. The microfluidic cartridge is therefore placed on a thermal actuation system associated with an optical module to record the real-time fluorescence. The quantification of the dumbbell using LAMP in this second architecture was possible down to 10 pM.
Our detection method using LAMP is generic and opens many perspectives for its application to other types of cardiac biomarkers (NTproBNP, miRNA…). The versatility of the microfluidic technology and the instrument developed should allow the multiplexed detection of cardiac biomarkers on this platform using this generic detection method, and should improve cardiovascular diseases diagnostic.

Keywords:
Point-of-Care, Microfluidic, Multiplex, Aptamers, Cardiac markers