AUTOMATED MICROFLUIDIC PLATFORM FOR ON FIELD HIGH SENSITIVITY QUANTIFICATION OF CARDIAC TROPONIN I
Résumé
Cardiovascular diseases are the leading cause of death in the world and several biomarkers are used clinically to diagnose patients [1]. Cardiac troponin is currently the gold standard for diagnostic of acute myocardial infarction (AMI), and in hospital emergency departments, testing patient blood for cardiac troponin is crucial to rule-in or rule-out patients [2]. Detecting troponin rapidly with high sensitivity and specificity is therefore of utmost importance, but current portable systems like Point-of-Care devices lack sensitivity [1]. We propose an on field innovative quantification assay integrated in a microfluidic cartridge on a fully automated transportable instrument for the high sensitivity sensing of cardiac troponin I. To improve the sensitivity of current molecular assays, we developed an original method based on LAMP amplification with the help of an original oligonucleotide structure. In our previous work, we developed this method with aptamers using thrombin protein as a model [3], and we now decline its use for high sensitivity troponin sensing. This innovative assay employs a protocol based on a few successive steps carried out in a versatile polymeric microfluidic cartridge placed on an automated and transportable instrument [4]. A dedicated cartridge architecture was specifically designed to integrate the troponin I detection protocol (Figure 1). Every step of the protocol is integrated on chip thanks to pneumatic actuation for fluid handling, magnetic separation, heating system and optical detection piloted via the instrument (Figure 2). The fluid handling system allows a precise volume calibration and the possibility to generate a calibration curve with spiked samples. Firstly, antibody coated magnetic beads are added to the sample inside the chip to specifically capture the troponin I target. Then, a sandwich complex is formed using a detection antibody that is combined with an oligonucleotide sequence [3]. The magnetic separation allows efficient washing steps on the magnetic beads. Lastly, exponential amplification of the detection probe will take place on chip thanks to a thermal actuation and an optical system recording the increase in fluorescence. Figure 3 shows results for troponin I detection in buffer medium on 4 orders of magnitude with a limit of detection in the picomolar range. This corresponds to the expected cTnI concentrations in the physiological and pathological ranges. We have demonstrated that combining antibody recognition for specific detection of troponin I and exponential amplification for sensitivity resulted in a low limit of detection and a wide dynamic range. Troponin I was successfully detected with this innovative ImmunoLAMP method and each step of the protocol was successfully integrated inside the microfluidic cartridge, with an appropriate architecture design, and its associated instrument. Current work is ongoing with the detection of cTnI in clinical blood samples and the complete integration of the LAMP amplification inside the microfluidic chip. This new method paves the way for other cardiac biomarkers quantifications. Indeed, LAMP amplification appears as a versatile method that can be applied to detect cardiac biomarkers presenting different biochemical natures (peptide, DNA, RNA). Moreover, the microfluidic technology developed here allows for the multiplexed detection of several biomarkers on a single platform. In this way, a fully automated Point-of- Care system detecting several cardiac biomarkers should improve cardiac pathologies diagnosis. References [1] M. Savonnet et al “Recent advances in cardiac biomarkers detection: From commercial devices to emerging technologies,” J. Pharm. Biomed. Anal., vol. 194, p. 113777, Feb. 2021, doi: 10.1016/j.jpba.2020.113777. [2] JP. Collet et al “2020 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation" European Heart Journal Volume 42, Issue 14, 7 April 2021, Pages 1289–1367 [3] M. Aubret et al “Development of an Innovative Quantification Assay Based on Aptamer Sandwich and Isothermal Dumbbell Exponential Amplification,” Anal. Chem., vol. 94, no. 7, pp. 3376–3385, Feb. 2022, doi: 10.1021/acs.analchem.1c05532. [4] C. Parent et al., “A versatile and automated microfluidic platform for a quantitative magnetic bead based protocol: application to gluten detection,” Lab. Chip, May 2022, doi: 10.1039/D2LC00328G.
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