Polymerization nicking-triggered LAMP cascades enable exceptional signal amplification for aptamer-based label-free detection of trace proteins in human serum

Abstract

Detecting molecular biomarkers in high sensitivity plays an important role in the diagnosis of various diseases at the early stage. Here, by combining the target-induced polymerization nicking reaction (TIPNR) with the loop-mediated isothermal amplification (LAMP), we describe an ultrasensitive and label-free aptamer-based sensing method for detecting low levels of proteins in human serum by using thrombin as the model target analyte. The target thrombin binds and causes spontaneous assembly of two distinct aptamer probes to form the templates for the polymerization nicking reaction recycling amplification to produce many forward inner primer sequences. Subsequently, downstream LAMP reactions are initiated by these sequences for the generation of tremendous DNA hairpins with various lengths via automated cyclic strand displacement reactions. The SYBR Green I organic dye further binds the many hairpins to show drastically amplified fluorescence for ultrasensitive detection of thrombin down to 3.6 fM in the linear range from 0.01 pM to 10 nM. Such a sensing method based on aptamers has high discrimination capability for the target molecules against other non-specific proteins and is applicable for diluted serum samples. With the successful demonstration of the substantial signal amplification ability and simplicity feature of this assay approach, highly sensitive and convenient detection of other disease biomarkers with this method can be envisioned in the near future.