Bladder cancer (BCa) is the 10th most often diagnosed cancer globally, known for its rapid progression and high recurrence rate (Dobruch and Oszczudlowski, 2021; Farling, 2017). Early diagnosis and monitoring for recurrence of BCa are crucial in clinical practice. Urinary exfoliation cytology is a frequently employed technique for identifying bladder cancer, however its clinical utility is restricted due to its low sensitivity (Ahmadi et al., 2021). It is crucial to identify a non-invasive, sensitive, and specific diagnostic biomarker for BCa promptly. Exosomes are small extracellular vesicles that transport cell-specific proteins, lipids, and nucleic acids and can be found in nearly all bodily fluids (Lai et al., 2022). Previous research has demonstrated that the quantity of exosomes and the biomolecules they transport are significant factors in the development and spread of BCa (Ng et al., 2021; Sugeeta et al., 2021; Tong et al., 2021). They are anticipated to serve as a crucial marker for the early detection of BCa. Thus, it is crucial to develop a non-invasive exosome detection platform relevant to breast cancer.
Exploring rapid and effective detection methods for exosomes has been motivated by their significance in medical research (He et al., 2018). Traditional immune-affinity-based methods including western blotting (Kowal et al., 2017), enzyme-linked immunosorbent assay (Khodashenas et al., 2019), and flow cytometry (Liu et al., 2022) have been utilized for exosome analysis but are constrained by certain limitations. Flow cytometry is expensive and relies significantly on instrument maintenance, while both western blotting and enzyme-linked immunosorbent assay have inherent sensitivity constraints. Recently, many assays targeting proteins have been created to address issues associated with traditional methods by directly identifying protein targets on exosome surfaces using fluorescence (Pan et al., 2020; Zhou et al., 2022), colorimetric (Kuang et al., 2022; Zhang et al., 2020), electrochemical (Kasetsirikul et al., 2022), and immunoassays (Li et al., 2022). Fluorescence-based approaches are particularly appealing due to their easy operation and the availability of commercially accessible fluorescent dyes. Xianxian Zhao et al. have created a fluorescence-based method for detecting exosomes (Zhao et al., 2020b). This method combines aptamer-based recognition of the CD63 protein on exosomes' surface with the trans-cleavage activity of the CRISPR-Cas12a system. They also suggested a way for detecting exosomes without the need for washing, using an allosteric probe and signal amplification mediated by the CRISPR-Cas12a system (Zhao et al., 2020a). Although fluorescent techniques have advanced significantly in detecting exosomes (Ibsen et al., 2017; Im et al., 2017), they only focus on individual protein molecules on the exosome surface, leading to inaccurate results in complex experimental settings. Hence, there is a strong need to create an innovative approach for detecting exosomes that can target several protein molecules on their surface.
Enzyme-linked immunosorbent assay is a traditional approach that can be expanded to detect several protein molecules, however it is often criticized for its limited sensitivity. We present a new approach for detecting exosomes that combines the immobilization of CD9 protein on exosomes' surface with aptamer-induced signal amplification, inspired by the enzyme-linked immunosorbent assay process. The antibody targets the CD9 protein, while the aptamer targets the CD63 protein, ensuring great precision. The dual cycle amplification includes the recycling of the exosomes-“recognizing probe” complex and the rolling circle amplification (RCA), giving the approach a higher sensitivity in comparison to the enzyme-linked immunosorbent test.