Label-Free Aptamer Probe For Activatable Detection Of Cancer Cells

As "chemical antibodies", aptamers have been drawn considerable attention in tumor diagnosis researches, due to the advantages of high affinity, excellent specificity, facile synthesis, easy modification and chemical stability. Currently, design of aptamer probes for tumor cell detection mainly relies on the "always on" strategy, which generally exhibits shortcomings such as high background, low sensitivity, complex operation and need of washing processes. In contrast, the activatable analysis strategy holds a great potential to improve sensitivity and simplify operations. However, the development of activatable aptamer probes is presently in its early stage. In particular, it is still urgently necessary to explore novel label-free activatable aptamer probes for tumor cell detection. In this thesis, aiming at the above frontier research direction, two kinds of novel label-free activatable aptamer probes have been constructed by using human leukemia CCRF-CEM cells as the study object, and the related tumor cell detection studies have been conducted.The detailed description is listed as follows:1. Label-free aptamer probe for activatable tumor cell detection based on binding-triggered catalysis of split DNAzymeBasing on the controllability of split DNAzyme’s catalysis activity and the advantage of aptamer’s flexible sequence design and variable conformations, a novel tailed hairpin-structured activatable aptamer probe (THAAP) was constructed by using a PEG linker to integrate tumor-specific aptamer and split G-quadruplex into one sequence. In the free state, the THAAP is hairpin-structured and the formation of G-quadruplex-hemin DNAzyme is inhibited, thus presenting a relatively low catalysis activity. However, as the target tumor cell is introduced, the binding of aptamer with the specific receptor in cell surface could effectively trigger the conformational alteration of THAAP, thus releasing the split G-quadruplex sequence. The split G-quadruplex sequence could then bind with hemin and form the peroxidase-llke structure, thus realizing the colorimetric detection of target tumor cells. By selecting the specific aptamer against CCRF-CEM cells as the model and using a Microplate Reader to acquiring colorimetric signals, a series of factors, including the composition and pH of buffer, the concentrations of hemin and substrates, and the sequence of THAAP, were optimized. And a label-free activatable aptamer probe was successfully constructed for the specific detection of CCRF-CEM cells with quantitative responses in the range of0-26,900cells and a lowest detection amount of3.3×103cells. In view of the expanding aptamer discovery for varying cancer targets, the THAAP design might hold a great potential to be developed as a simple, fast, specific, label-free and versatile platform for activatable tumor cell analysis.2. DNA-templated silver nanocluster-aptamer probe for activatable tumor cell detection based on intracellular fluorescence enhancement effectA novel DNA-templated silver nanocluster probe (Ag NCs) with intracellular fluorescence enhancement effect has been constructed based on the fluorescence enhancement feature of Ag NCs in proximity of G-rich DNA and the abundance of G-rich sequences in telomere of cell nuclei. By using flow cytometry and selecting fixed tumor cells as the model, a series of influence factors were investigated including incubation time, incubation concentration, and the sequence of DNA templates. Results showed that the intracellular fluorescence enhancement effect was obviously time-dependent and concentration-dependent. With the extension of incubation time and the increase of incubation concentration, the fluorescence signal of tumor cells was found to gradually enhance. Moreover, the attachment of a random DNA sequence on the DNA template was detected to be able to promote the fluorescence enhancement effect. And with the sequence length increased, the fluorescence signal firstly increased and then decreased. It was also revealed through laser confocal microscopy that the activated Ag NCs mainly located in cell nuclei. On this basis, the specific recognition of Sgc8c to CCRF-CEM cells was further combined with Ag NCs as the activatable signal generation element. And two kinds of Ag NCs-aptamer probes were constructed by employing the one-sequence integration design and the two-sequence hybridization design respectively, which successfully achieved the specific activatable detection of target cells. These results lay a foundation for the development of a simple, rapid, sensitive, specific and label-free activatable tumor cell detection method.