Development of an innovative quantification assay based on aptamer sandwich and isothermal dumbbell exponential amplification

Detecting blood biomarkers such as proteins with high sensitivity and specificity is of utmost importance for early and precise diseases diagnosis [1]. As molecular probes, aptamers are raising an increasing interest for biosensors applications as a replacement for antibodies which are used in classical enzyme-linked immuno-assays (ELISA) [2]. By combining the specificity of aptamers as molecular probes with the sensitivity of the isothermal loop mediated amplification (LAMP), we describe a sensitive and antibody-free molecular quantification method. For the proof-of-concept, we considered the thrombin involved in the complex coagulation cascade as a model protein [3] for which two aptamers have been selected and form a stable sandwich to capture the protein [4]. This assay employs a protocol based on few successive steps, similarly to ELISA. First, aptamer coated magnetic beads are added to the sample to specifically capture the targets. Then, a sandwich complex formation is performed using the second aptamer. The aptamer sequence is integrated in a larger oligonucleotide sequence designed to be amplified by LAMP with only two primers. After a proper rinsing step, the isothermal and exponential amplification allows us to detect and quantify low amount of targets (LOD ~100 pM) in complex media such as serum. This study demonstrates that our innovative biosensor based on aptamer sandwich and isothermal dumbbell exponential amplification allows for the detection of physiological thrombin concentrations and its quantification. This innovative and highly sensitivity assay could open the way for various applications of antibody-free molecular assays. References: [1] G.-J. Zhang, Z. H. H. Luo, M. J. Huang, J. J. Ang, T. G. Kang, and H. Ji, “An integrated chip for rapid, sensitive, and multiplexed detection of cardiac biomarkers from fingerprick blood,” Biosensors and Bioelectronics, vol. 28, no. 1, pp. 459–463, Oct. 2011, doi: 10.1016/j.bios.2011.07.007. [2] Y. Wu, I. Belmonte, K. S. Sykes, Y. Xiao, and R. J. White, “Perspective on the Future Role of Aptamers in Analytical Chemistry,” Anal. Chem., vol. 91, no. 24, pp. 15335–15344, Dec. 2019, doi: 10.1021/acs.analchem.9b03853. [3] C. Daniel, Y. Roupioz, T. Livache, and A. Buhot, “On the use of aptamer microarrays as a platform for the exploration of human prothrombin/thrombin conversion,” Analytical Biochemistry, vol. 473, pp. 66–71, Mar. 2015, doi: 10.1016/j.ab.2014.12.015. [4] C. Daniel, F. Mélaïne, Y. Roupioz, T. Livache, and A. Buhot, “Real time monitoring of thrombin interactions with its aptamers: Insights into the sandwich complex formation,” Biosensors and Bioelectronics, vol. 40, no. 1, pp. 186–192, Feb. 2013, doi: 10.1016/j.bios.2012.07.016.