Light-up aptamers have attracted growing attention due to their advantages of being label-free and having low fluorescence background. In this work, we developed a light-up fluorescence assay for label-free detection of tumor cells based on a bifunctional split aptamer (BFSA) that contained two DNA strands (BFSA-a and BFSA-b). BFSA-a and BFSA-b were constructed by combining aptamers ZY11 and ThT.2-2, which could specifically bind to the tumor cell SMMC-7721 and activate the fluorescence of thioflavin T (ThT). A Helper strand was introduced to hybridize with BFSA-b, and then BFSA-a and BFSA-b were separated if the target cell was absent. Only when the target cell is present can BFSA-a approach and hybridize with BFSA-b due to the 'induced-fit effect', which made the Helper strand dissociate. Then ThT bound to BFSA and the fluorescence of ThT was activated. The results indicated that this fluorescence assay had a good linear response to the target cells in the range of 250-20 000 cells in 100 μL binding buffer; the lowest cell number actually detected was 125 cells in 100 μL buffer. This assay also displayed excellent selectivity and was successfully applied to detect target cells in 20% human serum samples. The design of bifunctional split aptamers realized no-washing, label-free, low-cost, one-step detection of tumor cells, which could generate detectable fluorescence signals just by mixing nucleic acid aptamers and fluorescent reporter molecules with target cells. Such a design of aptamer probes also has the potential to construct stimuli-responsive controlled drug delivery systems.

Antibodies are the most common affinity reagents for specific target recognition. However, their applications are limited by high cost and low stability. Thus, seeking substitutes for antibodies is of great significance. In this work, we designed a library containing 82 self-assembled nanoparticles (SNPs) based on the self-assembly of β-cyclodextrin polymers and adamantane derivatives, and then screened out eight types of SNPs capable of suppressing the toxicity of melittin using a hemolytic activity neutralization assay. The affinities of the SNPs to melittin were demonstrated using surface plasmon resonance (SPR). As evidenced by cytotoxicity experiments, SNPs could also suppress the toxicity of melittin to other cells. In addition, to verify the universality of our method, 11 types of SNPs capable of neutralizing another toxic peptide, phenolic soluble polypeptide (PSMα3) secreted by Staphylococcus aureus, were selected from the same SNP library. Our self-assembly-based method for the library preparation has the advantages of flexible design, mild experimental condition, and simple operation, which is expected to seek artificial affinity reagents for more species.

No abstract