The accurate detection of immune checkpoint molecules on the surface of tumor cells is of great significance for immunotherapy. Herein, we propose a method based on cellular interface supported cascade signal amplification mediated by CRISPR/Cas13a system for in situ analysis of immune checkpoint molecules on bladder cancer cells. CD47 and EpCAM on bladder cancer cells can be sensitively monitored through T7 transcription amplification, CRISPR/Cas13a system and HCR triple signal amplification. Moreover, the established method can specifically differentiate EpCAM and CD47 positive bladder cancer cells and well serve for the evaluation of two kinds of membrane protein inhibitors. In a word, the method we proposed can successfully realize in situ analysis of immune checkpoint molecules on bladder cancer cells for guiding immunotherapy.
Single-atom nanozyme (SAZ) with peroxidase-like activity has attracted great attention in point-of-care (POC) diagnostic, but the use of unstable H 2 O 2 in peroxidase catalytic reactions often leads to low detection accuracy. Thus, developing SAZ with high oxidase (OD)-like activity to construct RNA sensing systems independent of H 2 O 2 is imperative for improving accuracy. Herein, a novel strategy to fabricate boron-nitrogen co-doped zinc SAZ (ZnBNC-SAZ) with excellent OD-like activity by carbonizing Zn based zeolitic boron imidazolate frameworks is reported. The electron deficient B in imidazolate ligand can form Zn─N─B bond in situ during high temperature pyrolysis, which can regulate the electronic structure of Zn and further upshift the d-band center of Zn to improve the OD-like activity. With ZnBNC-SAZ as signal generator, a new method for sensitive RNA detection is developed by combining CRISPR/ cas13 system with terminal deoxynucleotidyl transferase-induced DNA extension reaction. The limits of detection for RNAs are as low as 20 aM. The proposed biosensor is adaptable to a lateral-flow-based readout and is universally applicable for sensing various RNAs by programming the guide RNA. Importantly, the proposed biosensor can monitor the cellular differentiation and identify patients with cervical carcinoma, showing great potential for application in facile POC diagnosis.
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