Novel autonomous protein-encoded aptamer nanomachines and isothermal exponential amplification for ultrasensitive fluorescence polarization sensing of small molecules

Abstract

We develop a new type of autonomous protein-encoded aptamer nanomachine for amplified fluorescence polarization (FP) sensing of small molecules in homogeneous solutions. A DNA hairpin carrying the aptamer sequence for the target and the aptamer sequence for an enhancer protein carcinoembryonic antigen (CEA) is bound with CEA, providing the skeleton of the functional nanomachine. Upon recognition and binding to the target, the stem of the DNA hairpin is opened. Subsequently, the opened DNA hairpin anneals with a fluorophore-labeled primer, leading to the recycling of the target (aided by DNA polymerase) and the continuous generation and recycling of DNA sequences (assisted by both DNA polymerase and nicking endonuclease) to achieve isothermal exponential amplification (EXPA). This EXPA results in the generation of numerous DNA–protein complexes with the fluorophore, generating a substantial increase of the FP value due to the relative large volume of these DNA–protein complexes. This provides a readout signal for the amplified sensing process. By employing a nanomachine-based experimental model with aflatoxin B1 (AFB1) as the analyte, we demonstrate selective detection of AFB1 with a detection limit of 0.24 pM. The method is flexible, and we also show ultrasensitive detection of cocaine by switching the corresponding aptamer sequence in the nanomachine; the detection limit for cocaine is 18 pM with a wide dynamic range of six orders of magnitude in log concentration, again with high assay selectivity. Furthermore, this nanomachine-based sensing platform is capable of detecting target molecules in complex samples. Considering the remarkable signal amplification and simple machine-like operation, the developed nanomachine can be expected to provide a general protocol for ultrasensitive analysis of a variety of target molecules.