Research Rationale

Breast cancer is one of the most prevalent malignant tumors in women worldwide with strong heterogeneity, and accurate early diagnosis is critical to its prognosis. Current clinical detection methods are limited by high invasiveness, cumbersome procedures and insufficient early-stage sensitivity, creating an urgent demand for highly sensitive and convenient early screening technologies. Existing studies have demonstrated that exosomes serve as vital targets for liquid biopsy. The surface proteins HER2, EpCAM and PD-L1 of exosomes are closely associated with the typing and progression of breast cancer, and corresponding nucleic acid aptamers are available for specific recognition. This project intends to construct an Aptamer-PfAgo fluorescent detection platform to provide a novel approach for early breast cancer screening.

Design Concept

This study aims to establish a novel method based on high-load magnetic bead-multivalent APT-PfAgo for the simultaneous detection of three core breast cancer biomarkers in plasma. In the presence of biomarkers, they bind to aptamers immobilized on magnetic beads and release complementary strands, converting protein signals into cleavable nucleic acid signals. The supernatant containing free complementary strands is collected via magnetic separation to activate the PfAgo system, and biomarkers are quantitatively detected according to fluorescence intensity. Reserved expansion interfaces are designed for compatibility with test strips and portable detectors to broaden application scenarios.

Experimental Content

The research is carried out focusing on exosome capture, molecular design, system optimization and performance evaluation. Firstly, exosome enrichment and biomarker validation are completed. Secondly, structural prediction, molecular docking and sequence design are performed for aptamers, APT-c, gDNA and fluorescent probes. Subsequently, conditions for magnetic bead coating and displacement reaction are optimized. Finally, the sensitivity, specificity and methodological feasibility of the assay are evaluated.

Experimental Materials

Aptamers, APT-c, gDNA and fluorescent probes targeting HER2, EpCAM and PD-L1 proteins; magnetic beads; PfAgo enzyme; MnCl₂; reaction buffer; standard products of HER2, EpCAM and PD-L1 proteins.

Preliminary Experimental Results

The optimization of exosome capture conditions and biomarker validation have been completed, with optimal coating parameters obtained. Structural analysis of aptamers and design of APT-c and gDNA have been finished, and the PfAgo fluorescent detection system has been established. The optimal magnetic bead APT coating condition is 37 °C for 30 min, with an average magnetic bead loading capacity of 196.52 nM, enabling efficient magnetic enrichment. The optimal condition for displacement reaction is 27.5 °C for 30 min. The linear detection range for the three proteins is 1 ng/mL–1 μg/mL, with the limit of detection (LOD) of EpCAM, HER2 and PD-L1 being 165 pg/mL, 158 pg/mL and 113 pg/mL respectively, showing favorable specificity.

Feasibility

A complete experimental workflow and parameter framework have been established, with all key procedures supported by preliminary experiments. All nucleic acid components required are chemically synthesizable, and the detection system possesses great potential for further standardization and commercialization.

Innovations

For the first time, aptamer recognition, magnetic bead separation and PfAgo cleavage technology are integrated to realize simultaneous detection of multiple exosomal biomarkers for breast cancer.

The platform integrates quantitative fluorescence detection and point-of-care testing (POCT), showing promising potential for on-site detection transformation.

Keywords: Breast cancer; Exosome; Aptamer; PfAgo; POCT

Zhang Shanfeng, male, is a senior experimentalist and master's supervisor. He is a member of the Microbiology Society of Henan Province, an active member of the American Association for Cancer Research (AACR), and a lifelong member of the Chinese Society of Biochemistry and Molecular Biology. He serves as an editorial board member for the Journal of Surgery and Implants and the Online Journal of Cardiology Research & Reports (OJCRR), and as a reviewer for Frontiers, Anti-Cancer Agents in Medicinal Chemistry, Current Chemical Biology, and Life Science and Technology. He has published more than 36 SCI papers with a total citation of 821. He has undertaken 5 projects funded by the National Natural Science Foundation of China, and has edited 4 textbooks and monographs. He has obtained 4 utility model patents. He has received the Second Prize of Scientific and Technological Progress in Henan Province and the Special Prize of Higher Education Teaching Achievement Award in Henan Province.

Memberships:

Microbiology Society of Henan Province

American Association for Cancer Research (AACR)

Chinese Society of Biochemistry and Molecular Biology

Journal of Surgery and Implants

Online Journal of Cardiology Research & Reports (OJCRR)

Frontiers

Anti-Cancer Agents in Medicinal Chemistry

Current Chemical Biology

Life Science and Technology

Publications:

More than 36 SCI papers with a total citation of 821

Research Projects:

Undertaken 5 projects funded by the National Natural Science Foundation of China

Editor of 4 textbooks and monographs

Obtained 4 utility model patents

Awards:

Second Prize of Scientific and Technological Progress in Henan Province

Special Prize of Higher Education Teaching Achievement Award in Henan Province