Colorimetric detection of proteins based on target-induced activation of aptazyme

The detection of protein is vital to fundamental research as well as practical applications. However, most detection methods depend on antibody-based assays which are faced with many shortcomings. Herein, we propose a colorimetric method for protein assays based on target-triggered activation of aptazyme, which may offer simple, rapid and cost-effective detection of the target protein. In this method, the conformation change of aptazyme induced by target protein is designed to be associated with aptazyme activation. Consequently, in the presence of the target protein, the designed DNA linkers will be cleaved into two fragments that fail to cross-link gold nanoparticles (GNPs), thus the color of GNP solution remains red, while the color will be changed in the absence of the target. Because of the advantages of aptazyme such as economic synthesis, stable, easy modification and its ability to accomplish signal recognition and signal amplification simultaneously, the method is thermostable, simple and cost-efficient. In this work, we have taken the detection of vascular endothelial growth factor (VEGF) as an example, which can present an analytical performance with as low as 0.1 nM detection limit, spanning a detection range of 3 orders of magnitude. What is more, the principle of this proposed new method can be extended as a universal assay method not only for the detection of analytes which have an aptamer but also for those analytes that have ligands.     

Inhibited aptazyme-based catalytic molecular beacon for amplified detection of adenosine

Combining the inhibited aptazyme and molecular beacon(MB),we developed a versatile sensing strategy for amplified detection of adenosine.In this strategy,the adenosine aptamer links to the 8-17 DNAzyme to form an aptazyme.A short sequence,denoted as inhibitor,is designed to form a duplex spanning the aptamer–DNAzyme junction,which blocks the catalytic function of the DNAzyme.Only in the presence of target adenosine,the aptamer binds to adenosine,thus the inhibitor dissociates from the aptamer portion of the aptazyme and can no longer form the stable duplex required to inhibit the catalytic activity of the aptazyme.The released DNAzyme domain will hybridize to the MB and catalyze the cleavage in the presence of Zn2+,making the fluorophore separate from the quencher and resulting in fluorescence signal.The results showed that the detection method has a dynamic range from 10 nmol/L to 1 nmol/L,with a detection limit of 10 nmol/L.     

Molecular recognition triggered aptazyme cascade for ultrasensitive detection of exosomes in clinical serum samples

Exosomes have attracted widespread interest due to their inherent advantages in tumor diagnosis and treatment monitoring. However, it is still a big challenge for highly sensitive and specific detection of exosome in real complexed samples. Herein, a molecular recognition triggered aptazyme cascade strategy was developed for ultrasensitive detection of cancer exosomes in clinical serum samples. In this design, one target exosome could capture a large quantity of aptazymes for the first-step signal amplification. And then the captured aptazyme was activated and recycled to release the fluorophore-labelled substrate strand for a cascaded signal amplification. Notably, the activation of aptazyme only occurs when it has bound with target exosome, ensuring a low background. The experimental results show that the limit of detection (LOD) and the limit of quantification (LOQ) are 3.5 × 10³ particles/μL and 1.7 × 10⁴ particles/μL, respectively, which is comparable to the results of most existed fluorescence-based exosome probes. Moreover, this assay possesses high specificity to distinguish exosomes derived from other cell lines. Furthermore, this fluorescence probe was utilized in cancer patient and healthy serum samples successfully, suggesting its great potential for clinical diagnosis and biological studies.