Experimentally selected single-stranded DNA and RNA aptamers are able to bind to specific target molecules with high affinity and specificity. Many analytical methods make use of affinity binding between the specific targets and their aptamers. In the development of these methods, thrombin is the most frequently used target molecule to demonstrate the proof-of-principle. This paper critically reviews more than one hundred assays that are based on aptamer binding to thrombin. This review focuses on homogeneous binding assays, electrochemical aptasensors, and affinity separation techniques. The emphasis of this review is placed on understanding the principles and unique features of the assays. The principles of most assays for thrombin are applicable to the determination of other molecular targets.

Nucleic acids can be programmed into enzyme-free catalytic DNA circuits (CDCs) to carry out various functions ranging from DNA computing to signal amplifications for biosensing. Catalytic hairpin assembly (CHA), the accelerated hybridization between two DNA hairpins catalyzed by a DNA input, is one of the most widely studied and used CDCs for amplified detection of nucleic acids and small molecules. So far, it is still challenging to expand CHAs to proteins largely due to the lack of a universal strategy to construct protein-responsive CHAs. To address this challenge, we demonstrate that a rationally designed protein-DNA binding complex can be used as an effective catalyst to accelerate CHA reactions. On the basis of this principle, we developed specific CHAs for a number of important protein biomarkers, including human α-thrombin, human prostate specific antigen, and human epidermal growth factor receptor 2. Upon establishing this panel of protein-responsive CHAs, we further explore their potential applications to the detection of specific protein biomarkers from human serum samples and cancer cells.

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DNA three-way junctions (DNA TWJs) are important building blocks to construct DNA architectures and dynamic assemblies. We describe here a binding-induced DNA TWJ strategy that is able to convert protein bindings to the formation of DNA TWJ. The binding-induced DNA TWJ makes use of two DNA motifs each conjugated to an affinity ligand. The binding of two affinity ligands to the target molecule triggers assembly of the DNA motifs and initiates the subsequent DNA strand displacement, resulting in a binding-induced TWJ. Real-time fluorescence monitoring of the binding-induced TWJ enables detection of the specific protein targets. A detection limit of 2.8 ng/mL was achieved for prostate-specific antigen. The binding-induced TWJ approach compares favorably with the toehold-mediated DNA strand-displacement, the associative (combinative) toehold-mediated DNA strand-displacement, and the binding-induced DNA strand-displacement. Importantly, the binding-induced TWJ broadens the scope of dynamic DNA assemblies and provides a new strategy to design protein-responsive DNA devices and assemblies.