Automated sample preparation microfluidic platform for immuno-LAMP detection: application to troponin I quantification.

Abstract

Easy-to-use, highly-sensitive, and quantitative assays over a large dynamic range are needed to improve patient care and disease monitoring. These factors are challenging to address simultaneously using classical methods. The laboratory ELISA assay is relevant for complex matrices over a linear quantification range, whereas immunoassay implementation in commercial lateral flow assays (LFA) remains mostly qualitative. In the case of myocardial infarction (MI), the cardiac biomarker troponin I (cTnI) is present at low levels in the blood of a healthy patient but increases by several orders of magnitude at the onset of symptoms. Here, we aim to detect cTnI as a proof of concept for developing a sample preparation microfluidic system for the immuno loop-mediated isothermal amplification (immuno-LAMP) method. Immuno-LAMP combines the sensitivity and selectivity of antibody recognition with the wide dynamic range allowed through nucleic acid amplifications. cTnI immuno-LAMP assays are firstly validated in a well-plate, and then optimized and integrated into microfluidic cartridges to improve reliability and enable automation. A network of valves and chambers controls a sample preparation module that handles magnetic beads coupled to capture antibodies. Detection antibodies coupled to short oligonucleotides, used to form the immuno-complex, hybridize with an oligonucleotide structure called a dumbbell. This structure is isothermally amplified in tubes to allow the quantification of cTnI. Our work has demonstrated the ability of our automated sample preparation platform to quantify cTnI spiked in human blood plasma at sub-picomolar concentrations. This prototype opens perspectives for integrating the whole immuno-LAMP method from human plasma samples in a microfluidic device including the LAMP amplification and detection.