Point-of-Care medical device development for high sensitivity multiplexed detection of cardiac biomarkers
Résumé
Cardiovascular diseases are the leading cause of death in the world, and several biomarkers have been validated and are now used clinically to diagnose patients with cardiovascular disease symptoms. In hospital emergency department, testing patient blood for these biomarkers is crucial to rule-in or rule-out patients. Designing a tool that is able to detect rapidly and sensibly these biomarkers is important to help clinicians to take decisions regarding patients’ health state. Portable and self-running devices allowing in-vitro diagnostics like Point-Of-Care (POC) devices tend to be an interesting method to accelerate and optimize patient care. Reducing the time and the limit of detection of these tests by developing high sensitivity assays in POC systems remain currently key points to be addressed. Instead of classical ELISA immuno-assays, we develop an innovative assay to quantify cardiac biomarkers in blood. This assay is based on a molecular sandwich between the target protein of interest and aptamer or antibody probes, followed by an original isothermal LAMP amplification in order to lower the assay limit of detection. Moreover, we aim to integrate this innovative aptamero- or immuno-LAMP assay in a microfluidic cartridge. This chip will carry out every step of the protocol, thus reducing sample volume and test time, and improving reproducibility and sensitivity. We also develop an automated instrument to handle all the operations achieved inside the cartridge in transportable device. An antibody-free molecular assay was validated using model targets (thrombin protein and short DNA), aptamer recognition and LAMP amplification to detect the targets on four orders of magnitude with a limit of detection in the nanomolar range. In case of cardiac biomarkers such as troponin or NTproBNP, the immuno-LAMP assay was considered, by combining antibody recognition and isothermal amplification. This method allowed us to detect the troponin I target on four orders of magnitude with a limit of detection in the picomolar range. We have demonstrated a proof of concept for a new sensing method that can be applied on several biomarkers to design multiplex cardiac assay and reach the required sensitivity. Combining this innovative assay with the microfluidic technology will allow us to reach lower limit of detection, and propose a multiplex assay carried out in a fully automated device.
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