Development of Dual-Aptamers for Constructing Sandwich-Type Pancreatic Polypeptide Assay

Pancreatic polypeptide (PP) is a specific biomarker of nonfunctional pancreatic neuroendocrine tumors (NF-pNETs). Clinical significance of PP inspires researchers to make great efforts in developing sensitive and specific sensors. However, there is no existing biosensor for detecting PP that combines facility and functionality. Addressing this challenge, a pair of aptamers which could be used to develop a sandwich assay for PP is reported. First, several high affinity aptamers are screened through graphene oxide-based SELEX, and appropriate dual-aptamers which could bind to different epitopes of PP are identified through fluorescence assays. Then the feasibility of the dual-aptamers for constructing the sandwich assay is validated via dynamic light scattering. This sandwich assay shows considerable sensitivity and specificity. The above results imply that the dual-aptamers have the potential toward developing novel sensors for PP in clinical samples

Evaluation of Medicine Effects on the Interaction of Myoglobin and Its Aptamer or Antibody Using Atomic Force Microscopy

The effects of medicine on the biomolecular interaction have been given increasing attention in biochemistry and affinity-based analytics since the environment in vivo is complex especially for the patients. Herein, myoglobin, a biomarker of acute myocardial infarction, was used as a model, and the medicine effects on the interactions of myoglobin/aptamer and myoglobin/antibody were systematically investigated using atomic force microscopy (AFM) for the first time. The results showed that the average binding force and the binding probability of myoglobin/aptamer almost remained unchanged after myoglobin-modified gold substrate was incubated with promazine, amoxicillin, aspirin, and sodium penicillin, respectively. These parameters were changed for myoglobin/antibody after the myoglobin-modified gold substrate was treated with these medicines. For promazine and amoxicillin, they resulted in the change of binding force distribution of myoglobin/antibody (i.e., from unimodal distribution to bimodal distribution) and the increase of binding probability; for aspirin, it only resulted in the change of the binding force distribution, and for sodium penicillin, it resulted in the increase of the average binding force and the binding probability. These results may be attributed to the different interaction modes and binding sites between myoglobin/aptamer and myoglobin/antibody, the different structures between aptamer and antibody, and the effects of medicines on the conformations of myoglobin. These findings could enrich our understanding of medicine effects on the interactions of aptamer and antibody to their target proteins. Moreover, this work will lay a good foundation for better research and extensive applications of biomolecular interaction, especially in the design of biosensors in complex systems

Selection of Aptamers for Hydrophobic Drug Docetaxel To Improve Its Solubility

With the development of combinatorial chemistry and high-throughput screening, the number of hydrophobic drug candidates continues to increase. However, the low solubility of hydrophobic drugs could induce erratic absorption patterns and affect the drug efficacy. Aptamers are artificially selected highly water-soluble oligonucleotides that bind to ions, small molecules, proteins, living cells, and even tissues. Herein, to increase the solubility of hydrophobic drug, we screened the aptamer by exploiting DNA library immobilization selection strategy and microfluidic technology. The highly water-soluble aptamer might influence the dissolving capacity of its target. To demonstrate the concept, docetaxel (DOC), a second-generation taxoid cytotoxic with significant antitumor agent activity, was chosen as the model. It is generally known that the clinical application of docetaxel is limited greatly owing to its poor water solubility and serious side effects. After seven rounds of selection, two docetaxel-specific aptamers DOC6–5 and DOC7–38, were successfully obtained, and their apparent dissociation constants (<i>K</i>_d) were at nanomolar level. Then these two 100 mer ssDNA aptamers against docetaxel were truncated to 22 mer ones by utilizing the recognition domain. Moreover, the shorter aptamer exhibited higher binding affinity than 100 mer ssDNA aptamers. By adding the optimized aptamer, the solubility of docetaxel was increased from ∼14 μM to ∼145 μM, and the cytotoxicity of docetaxel did not reduce in the presence of aptamer. Therefore, the aptamer was used as a solubilizer to improve the solubility of hydrophobic drug (docetaxel) in aqueous phase. This strategy may also be extended to other hydrophobic drugs. Meanwhile, this work could also provide a useful tool for tumor targeting therapy by combining with cell target ligands