volume | PIER Journals

Abstract

Exosomal surface proteins are emerging as minimally invasive liquid-biopsy biomarkers for breast cancer, yet their trace-level abundance poses challenges for biosensor sensitivity and multiplexing capacity, and conventional assays miss the substantial cargo in vesicles. Here, we report a mass-amplification-enhanced multiplexed SPR platform coupling antibody-directed magnetic enrichment with a phase-interrogated Ag/ZnSe waveguide chip for simultaneous detection of EpCAM, VEGF, and CD63, with a proof-of-concept extension to the immune-activation marker IFN-γ. A bimetallic Zr/Ce-MOF@Fe₃O₄ nanocomposite (confirmed by SEM-EDS) provided high refractive-index mass loading and magnetic separability, interfaced with a multi-channel Ag/ZnSe/APTES/GO chip (anti-EpCAM, anti-VEGF, anti-CD63) via a phase-polarization-modulation SPR system and PDMS microfluidics. MOF amplification improved the LOD for EpCAM from 0.86 to 0.07 pg mL^-1 (12.3-fold) and for VEGF from 2.45 to 0.21 pg mL^-1 (11.7-fold). Intact exosomes gave antibody-specific responses on anti-EpCAM (6.22°), anti-VEGF (3.09°), and anti-CD63 (9.54°), with signal-to-background ratios up to 106.0 and cross-reactivity below 0.4°; in matched healthy and patient serum, MOF-amplified responses were consistently higher in patients across all three markers (6.5-fold EpCAM, 6.2-fold VEGF, 8-fold CD63), with comparable discrimination extended to urine on the CD63 channel. Lysates analyzed on anti-EpCAM channels showed negligible healthy signals (0.29° without MOF, 1.63° with MOF) versus a patient increase from 5.80° to 18.4° (3.17-fold), corresponding to an apparent 41 pg mL^-1 total solubilized EV-associated EpCAM. As proof of concept for immune-status phenotyping, CD63-captured exosomes from a patient with clinical suspicion of immune activation showed a 12-fold higher IFN-γ signal than healthy samples (9-fold with MOF), with Langmuir kinetics indicating higher apparent binding affinity in the patient. Nanoparticle tracking confirmed exosome populations within the characteristic 30-150 nm range (mean 66.5-92.8 nm), and inter-chip/inter-channel reproducibility were 2.72% and 0.64% CV. As a complementary label-free imaging readout, the EV/MOF workflow was also implemented on a differential guided-mode resonance (dGMR) platform, where intact whole-EV binding produced MOF-amplified resonance-stripe pixel shifts without vesicle lysis; patient-derived EVs showed ~9.4-fold enhancement (vs 6.86-fold by phase-interrogated SPR), and healthy-derived EVs showed $\sim10.8$-fold enhancement, pushing the dGMR whole-EV response into a near-order-of-magnitude amplification regime. In a comparative discussion, sandwich SERS detection of EV-associated IFN-γ in CAR-T therapy further demonstrated that molecular fingerprint-based SERS can resolve specific immune markers, complementing the quantitative strengths of SPR and extending the platform toward immune-status monitoring. This platform demonstrates proof-of-concept feasibility for multiplexed exosomal marker detection directly in crude clinical matrices, with larger cohort studies required to establish diagnostic utility.

1. Kalluri, R. and V. S. LeBleu, "The biology, function, and biomedical applications of exosomes," Science, Vol. 367, No. 6478, eaau6977, 2020.
doi:10.1126/science.aau6977 Google Scholar

2. Xu, Rong, Alin Rai, Maoshan Chen, Wittaya Suwakulsiri, David W. Greening, and Richard J. Simpson, "Extracellular vesicles in cancer --- Implications for future improvements in cancer care," Nature Reviews Clinical Oncology, Vol. 15, No. 10, 617-638, May 2018.
doi:10.1038/s41571-018-0036-9 Google Scholar

3. Lin, Huixian, Bo Li, Jingyun Guo, Xueying Mai, Haiyang Yu, Weilun Pan, Bodeng Wu, Wei Liu, Mingzhen Zhong, Tong Liao, Ye Zhang, Bo Situ, Xiaohui Yan, Yifan Liu, Chunchen Liu, and Lei Zheng, "Simultaneous detection of membrane protein and mRNA at single extracellular vesicle level by droplet microfluidics for cancer diagnosis," Journal of Advanced Research, Vol. 73, 645-657, July 2025.
doi:10.1016/j.jare.2024.08.026 Google Scholar

4. Liu, Peng, Qun Zhang, and Fengchao Liu, "Biological roles and clinical applications of EpCAM in HCC," Discover Oncology, Vol. 16, No. 1, March 2025.
doi:10.1007/s12672-025-02095-0 Google Scholar

5. Amrollahi, Pouya, Meryl Rodrigues, Christopher J. Lyon, Ajay Goel, Haiyong Han, and Tony Y. Hu, "Ultra-sensitive automated profiling of EpCAM expression on tumor-derived extracellular vesicles," Frontiers in Genetics, Vol. 10, 1273, December 2019.
doi:10.3389/fgene.2019.01273 Google Scholar

6. Ko, Song Yi, WonJae Lee, Hilary A. Kenny, Long H. Dang, Lee M. Ellis, Eric Jonasch, Ernst Lengyel, and Honami Naora, "Cancer-derived small extracellular vesicles promote angiogenesis by heparin-bound, bevacizumab-insensitive VEGF, independent of vesicle uptake," Communications Biology, Vol. 2, No. 1, 386, October 2019.
doi:10.1038/s42003-019-0609-x Google Scholar

7. Moiseeva, Ekaterina, Igor Sergeev, Vasiliy Chernyshev, Olga Zaborova, Daria Kohzevnikova, Alexander Yakovlev, Olesya Kuznetsova, Alexey Tryakin, Aleksei Komlev, Dmitry Gorin, and Alexey Yashchenok, "Profiling of CD63 and EpCAM membrane proteins of extracellular vesicles on tannic acid-coated magnetic beads using conventional flow cytometry," International Journal of Molecular Sciences, Vol. 26, No. 23, 11324, November 2025.
doi:10.3390/ijms262311324 Google Scholar

8. Zhang, Haiying, Daniela Freitas, Han Sang Kim, Kristina Fabijanic, Zhong Li, Haiyan Chen, Milica Tesic Mark, Henrik Molina, Alberto Benito Martin, Linda Bojmar, Justin Fang, Sham Rampersaud, Ayuko Hoshino, Irina Matei, Candia M. Kenific, Miho Nakajima, Anders Peter Mutvei, Pasquale Sansone, Weston Buehring, Huajuan Wang, Juan Pablo Jimenez, Leona Cohen-Gould, Navid Paknejad, Matthew Brendel, Katia Manova-Todorova, Ana Magalhães, José Alexandre Ferreira, Hugo Osório, André M. Silva, Ashish Massey, Juan R. Cubillos-Ruiz, Giuseppe Galletti, Paraskevi Giannakakou, Ana Maria Cuervo, John Blenis, Robert Schwartz, Mary Sue Brady, Héctor Peinado, Jacqueline Bromberg, Hiroshi Matsui, Celso A. Reis, and David Lyden, "Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation," Nature Cell Biology, Vol. 20, No. 3, 332-343, February 2018.
doi:10.1038/s41556-018-0040-4 Google Scholar

9. Bandini, Erika, Tania Rossi, Emanuela Scarpi, Giulia Gallerani, Ivan Vannini, Samanta Salvi, Irene Azzali, Mattia Melloni, Sara Salucci, Michela Battistelli, Patrizia Serra, Roberta Maltoni, William C. Cho, and Francesco Fabbri, "Early detection and investigation of extracellular vesicles biomarkers in breast cancer," Frontiers in Molecular Biosciences, Vol. 8, 732900, November 2021.
doi:10.3389/fmolb.2021.732900 Google Scholar

10. Graham, Rosalind, Patrycja Gazinska, Birong Zhang, Atousa Khiabany, Shubhankar Sinha, Thanussuyah Alaguthurai, Fabian Flores-Borja, Jose Vicencio, Fabienne Beuron, Ioannis Roxanis, Rafal Matkowski, Revadee Liam-Or, Andrew Tutt, Tony Ng, Khuloud T. Al-Jamal, You Zhou, and Sheeba Irshad, "Serum-derived extracellular vesicles from breast cancer patients contribute to differential regulation of T-cell-mediated immune-escape mechanisms in breast cancer subtypes," Frontiers in Immunology, Vol. 14, 1204224, June 2023.
doi:10.3389/fimmu.2023.1204224 Google Scholar

11. Kong, Fang, Megha Upadya, Andrew See Weng Wong, Rinkoo Dalan, and Ming Dao, "Isolating small extracellular vesicles from small volumes of blood plasma using size exclusion chromatography and density gradient ultracentrifugation: A comparative study," eLife, RP92796, January 2024.
doi:10.7554/elife.92796.1 Google Scholar

12. Zhou, Yan, Guangyao Wu, Lili Cong, Xin Wang, Jiaqi Wang, Lianghai Hu, and Shuping Xu, "SERS analysis of cancer cell-derived exosome phenotypes using a rare-earth ion coordination-dominated exosome capture strategy," Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, Vol. 360, 128033, November 2026.
doi:10.1016/j.saa.2026.128033 Google Scholar

13. Chinnappan, Raja, Qasem Ramadan, and Mohammed Zourob, "Isolation and detection of exosomal Mir210 using carbon nanomaterial-coated magnetic beads," Journal of Functional Biomaterials, Vol. 14, No. 9, 441, August 2023.
doi:10.3390/jfb14090441 Google Scholar

14. Homola, Jiří, "Surface plasmon resonance sensors for detection of chemical and biological species," Chemical Reviews, Vol. 108, No. 2, 462-493, January 2008.
doi:10.1021/cr068107d Google Scholar

15. Kamal Eddin, Faten Bashar, Yap Wing Fen, Josephine Ying Chyi Liew, Nurul Illya Muhamad Fauzi, Wan Mohd Ebtisyam Mustaqim Mohd Daniyal, and Huda Abdullah, "Development of plasmonic-based sensor for highly sensitive and selective detection of dopamine," Optics & Laser Technology, Vol. 161, 109221, June 2023.
doi:10.1016/j.optlastec.2023.109221 Google Scholar

16. Daniyal, Wan Mohd Ebtisyam Mustaqim Mohd, Yap Wing Fen, Faten Bashar Kamal Eddin, Jaafar Abdullah, and Mohd Adzir Mahdi, "Surface plasmon resonance assisted optical characterization of nickel ion solution and nanocrystalline cellulose-graphene oxide thin film for sensitivity enhancement analysis," Physica B: Condensed Matter, Vol. 646, 414292, December 2022.
doi:10.1016/j.physb.2022.414292 Google Scholar

17. Yin, Zhiyong, JianPeng Wang, Xinlu Zhang, Xili Jing, and Shuguang Li, "Triple-channel SPR ion-imprinted fiber sensor enabled by MoS₂ thickness-tuned resonance for multiplexed heavy metal ion detection," Optics Letters, Vol. 50, No. 24, 7596-7599, December 2025.
doi:10.1364/ol.582326 Google Scholar

18. Liu, Zhenchao, Tingbiao Guo, Qin Tan, Houxin Fan, and Sailing He, "Guided mode induced surface phase mutation for enhanced SPR biosensor with dual-parameters interrogation," Advanced Materials Interfaces, Vol. 10, No. 34, 2300354, August 2023.
doi:10.1002/admi.202300354 Google Scholar

19. Yang, Kang, Yan Chen, Sen Yan, and Wenxing Yang, "Nanostructured surface plasmon resonance sensors: Toward narrow linewidths," Heliyon, Vol. 9, No. 6, e16598, June 2023.
doi:10.1016/j.heliyon.2023.e16598 Google Scholar

20. Kamal Eddin, Faten Bashar, Jian Sun, Guanghui Chen, Maofa Zeng, Wenjun Jin, Houxin Fan, De-Man Han, and Sailing He, "A phase-interrogated surface plasmon resonance sensor based on a graphene oxide-functionalized Ag/ZnSe platform for dopamine detection," Progress In Electromagnetics Research M, Vol. 138, 97-115, 2026.
doi:10.2528/pierm26060209 Google Scholar

21. Liu, Hezhen, Zhaofei Liu, Kwangnak Koh, and Hongxia Chen, "Magnetic enrichment and enabled cascade amplification for ultrasensitive SPR analysis of PD-L1+ exosomes," Biosensors and Bioelectronics, Vol. 311, 118920, November 2026.
doi:10.1016/j.bios.2026.118920 Google Scholar

22. Wang, Hongqiang, Jiayu Sun, Qingqing Zou, Bin Du, Hui Liu, Yanan Luan, Xin Wang, Xiaohai Yang, Qing Wang, and Kemin Wang, "Wash-free isolation and quantification of tumor-derived exosomes via In situ-formed hydrogel," ACS Sensors, Vol. 10, No. 6, 4136-4144, 2025.
doi:10.1021/acssensors.5c00204 Google Scholar

23. Tan, Wenqiao, Cuiling Zhang, Shasha Cheng, Xinyu Hu, Man Wang, and Yuezhong Xian, "DNA gate-based CRISPR-cas exponential amplification system for ultrasensitive small extracellular vesicle detection to enhance breast cancer diagnosis," Analytical Chemistry, Vol. 96, No. 3, 1328-1335, January 2024.
doi:10.1021/acs.analchem.3c04873 Google Scholar

24. An, Yu, Rui Li, Fan Zhang, and Pingang He, "Magneto-mediated electrochemical sensor for simultaneous analysis of breast cancer exosomal proteins," Analytical Chemistry, Vol. 92, No. 7, 5404-5410, 2020.
doi:10.1021/acs.analchem.0c00106 Google Scholar

25. Zhang, Mingwan, Ling Xia, Wenjing Mei, Qingqing Zou, Hui Liu, Hongqiang Wang, Liyuan Zou, Qing Wang, Xiaohai Yang, and Kemin Wang, "One-step multiplex analysis of breast cancer exosomes using an electrochemical strategy assisted by gold nanoparticles," Analytica Chimica Acta, Vol. 1254, 341130, May 2023.
doi:10.1016/j.aca.2023.341130 Google Scholar

26. Yildizhan, Yagmur, Venkata Suresh Vajrala, Edward Geeurickx, Charles Declerck, Nevena Duskunovic, Delphine De Sutter, Sam Noppen, Filip Delport, Dominique Schols, Johannes V. Swinnen, Sven Eyckerman, An Hendrix, Jeroen Lammertyn, and Dragana Spasic, "FO-SPR biosensor calibrated with recombinant extracellular vesicles enables specific and sensitive detection directly in complex matrices," Journal of Extracellular Vesicles, Vol. 10, No. 4, e12059, February 2021.
doi:10.1002/jev2.12059 Google Scholar

27. Zhang, Peng, Mei He, and Yong Zeng, "Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating," Lab on a Chip, Vol. 16, No. 16, 3033-3042, 2016.
doi:10.1039/c6lc00279j Google Scholar

28. Xu, Xun, Yuanyue Zhang, Muxue Wang, and Youchun Xu, "Digital quantification of EV proteins and miRNAs enables early cancer diagnosis with 10 μL of plasma," Chemical Engineering Journal, Vol. 522, 168310, October 2025.
doi:10.1016/j.cej.2025.168310 Google Scholar

29. Chen, Long, Shixin Zhai, Lisha Yang, Kai Chen, Zhuo Li, Tong Yu, Haijiao Xie, and Hongtao Zhu, "Self-enhancement of phosphorus recovery from anaerobic fermented sludge with MOF-808 synthesized with an improved green method," Chemical Engineering Journal, Vol. 479, 147880, January 2024.
doi:10.1016/j.cej.2023.147880 Google Scholar

30. Qian, Lei, Jun Cheng, Kai Xin, Hao Guo, Yuxiang Mao, Jiacan Tu, and Weijuan Yang, "Enhanced hydroxyl bridge-mediated microalgal lipid conversion via mixed-valence Zr/Ce-MOF-808 catalysts at reduced temperatures," Sustainable Energy & Fuels, Vol. 8, No. 15, 3304-3316, 2024.
doi:10.1039/d4se00647j Google Scholar

31. Zhu, Qi-Long and Qiang Xu, "Metal-organic framework composites," Chemical Reviews, Vol. 43, No. 16, 5468-5512, August 2014.
doi:10.1039/c3cs60472a Google Scholar

32. Türker, Argun and Orhan Acar, "Magnetic solid-phase extraction of lead(II) from water, tea, and linden leaf samples using a zirconium-based metal organic framework (Zr-MOF@Fe₃O₄) prior to determination by flame atomic absorption spectrometry," Food Analytical Methods, Vol. 18, No. 9, 2106-2119, July 2025.
doi:10.1007/s12161-025-02849-0 Google Scholar

33. Gonzalez-Villasana, Vianey, Mohammed H. Rashed, Yessica Gonzalez-Cantú, Recep Bayraktar, Jorge Luis Menchaca-Arredondo, Jose Manuel Vazquez-Guillen, Cristina Rodriguez-Padilla, Gabriel Lopez-Berestein, and Diana Resendez-Perez, "Presence of circulating miR‐145, miR‐155, and miR‐382 in exosomes isolated from serum of breast cancer patients and healthy donors," Disease Markers, Vol. 2019, 6852917, February 2019.
doi:10.1155/2019/6852917 Google Scholar

34. Tian, Fei, Shaohua Zhang, Chao Liu, Ziwei Han, Yuan Liu, Jinqi Deng, Yike Li, Xia Wu, Lili Cai, Lili Qin, Qinghua Chen, Yang Yuan, Yi Liu, Yulong Cong, Baoquan Ding, Zefei Jiang, and Jiashu Sun, "Protein analysis of extracellular vesicles to monitor and predict therapeutic response in metastatic breast cancer," Nature Communications, Vol. 12, No. 1, 2536, May 2021.
doi:10.1038/s41467-021-22913-7 Google Scholar

35. Yang, Xiaolin, Lining Liu, Yanlong Feng, Xuan Guo, Yang Wu, Qiang Gao, Chengxiao Zhang, and Honglan Qi, "Automatic electrochemiluminescence method for the detection of cancerous exosomes incorporating specific aptamer-magnetic beads and signal nanoprobes," Analytical Chemistry, Vol. 96, No. 25, 10459-10466, June 2024.
doi:10.1021/acs.analchem.4c01938 Google Scholar

36. Heidarzadeh, Morteza, Amir Zarebkohan, Reza Rahbarghazi, and Emel Sokullu, "Protein corona and exosomes: New challenges and prospects," Cell Communication and Signaling, Vol. 21, No. 1, 64, March 2023.
doi:10.1186/s12964-023-01089-1 Google Scholar

37. Liu, Zhenchao, Houxin Fan, Tingbiao Guo, Qin Tan, Zhi Zhang, Yuwei Sun, Julian Evans, Junbo Liang, Ruili Zhang, and Sailing He, "Ultrasensitive imaging-based sensor unlocked by differential guided-mode resonance," Nature Communications, Vol. 16, 6113, 2025.
doi:10.1038/s41467-025-60947-3 Google Scholar

Prof. Sailing He

Dr. Faten Bashar Kamal Eddin

Dr. Alamgir

Dr. Houxin Fan

Mr. Kaixin Zheng

Dr. Shuang E

Dr. Junbo Liang

Dr. Junbo Cai

Dr. Bojian Xie

Dr. Jinhua Ding

Dr. Mohammed Zourob

Dr. Hongsheng Lu

Dr. De-Man Han

Dr. Wenda Luo