Analytica chimica acta · 2025
Immunofluorescent analysis of exosomes using a microchip filled with transparent antibody-conjugated beads for breast cancer liquid biopsy.
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Li Na, Cheng Cheng, Wu Di, Song Zixuan, Wang Bei, Li Guiying, Yang Fang
Аннотация
BACKGROUND: Exosomes represent optimal biomarkers for liquid biopsy. Current exosome-based methods are still limited by isolation methods, long processing times, complex structure, low accuracy, large sample volume consumption, and the influence of chemical reagents on the analysis results.
RESULT: To overcome these challenges, a novel microfluidic approach that employs continuous, rapid, and efficient processes is proposed for the capture and enriching exosomes using the optical transparent antibody-conjugated microbeads within the chip. By interfering with laminar flow, increasing mass transfer efficiency, expanding the specific reaction area, and concentrating the fluorescent signal, the microbeads raise the sensitivity of the detection. Furthermore, multiple biomarkers of a single disease could be detected simultaneously by multi-color labeling with a single sample injection. This method required only 50 μL of plasma sample for detection within 35 min, demonstrating a low detection limit of 2.61 × 103 exosomes/μL, coupled with high reproducibility, stability, and specificity. We evaluated the diagnostic potential for breast cancer (BC) by analyzing plasma exosomes in samples from 15 patients with BC and 15 healthy individuals. The identification of total exosomes, EpCAM-positive exosomes, and MUC1-positive exosomes demonstrated a strong ability to distinguish between cancer patients and healthy individuals, as indicated by area under curve (AUC) values of 0.88, 0.98, and 0.99, respectively. The integrated multi-biomarker detection strategy achieved the highest of diagnostic accuracy and specificity (SUM, AUC = 1.0).
SIGNIFICANCE: This study presents a novel exosome-based liquid biopsy strategy, which is expected to enhance the accuracy and efficiency of early diagnosis for cancer and other diseases.
RESULT: To overcome these challenges, a novel microfluidic approach that employs continuous, rapid, and efficient processes is proposed for the capture and enriching exosomes using the optical transparent antibody-conjugated microbeads within the chip. By interfering with laminar flow, increasing mass transfer efficiency, expanding the specific reaction area, and concentrating the fluorescent signal, the microbeads raise the sensitivity of the detection. Furthermore, multiple biomarkers of a single disease could be detected simultaneously by multi-color labeling with a single sample injection. This method required only 50 μL of plasma sample for detection within 35 min, demonstrating a low detection limit of 2.61 × 103 exosomes/μL, coupled with high reproducibility, stability, and specificity. We evaluated the diagnostic potential for breast cancer (BC) by analyzing plasma exosomes in samples from 15 patients with BC and 15 healthy individuals. The identification of total exosomes, EpCAM-positive exosomes, and MUC1-positive exosomes demonstrated a strong ability to distinguish between cancer patients and healthy individuals, as indicated by area under curve (AUC) values of 0.88, 0.98, and 0.99, respectively. The integrated multi-biomarker detection strategy achieved the highest of diagnostic accuracy and specificity (SUM, AUC = 1.0).
SIGNIFICANCE: This study presents a novel exosome-based liquid biopsy strategy, which is expected to enhance the accuracy and efficiency of early diagnosis for cancer and other diseases.