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ultracentrifugation/magnetic bead methods 140 . Dorayappan et al. 141 developed a

microfluidic device combining anti-CD63 and anti-EpCAM antibodies for high-purity,

high-yield specific exosome isolation from culture media/patient serum.
(3) Low-Abundance Protein Biomarker Isolation

Detection of proteins and their metabolites in microfluidics is among the most

mature and commercialized microfluidics applications 142 . Breakthroughs exist in

integration, multiplexing, sensitivity, and throughput. Integrated devices perform

plasma separation from a blood drop while quantifying multiple proteins, matching

conventional ELISA efficiency. Emde et al. 143 used 3D printing to develop a chip

detecting APL-specific biomarkers (PML::RARA fusion protein), significantly

shortening development cycles for rapid prototyping/personalized diagnostics (Figure

7D). Sharafeldin et al. 144 designed and 3D-printed a low-cost microfluidic

immunoarray for on-chip cell lysis and quantitative protein analysis. Antibodies

captured in chambers coated with swellable 3D chitosan hydrogel films enabled

sandwich immunosorbent assays, successfully detecting metastatic head and neck

squamous cell carcinoma markers like desmoglein 3 (DSG3) and co-biomarkers.
Overall, these integrated systems utilize continuous perfusion of whole blood

through cascaded microchambers and microchannels that sequentially apply multiple

microscale separation principles, including deterministic lateral displacement (DLD),

inertial focusing, affinity capture, and size-exclusion filtration, to achieve high-purity

isolation of diverse blood components such as leukocytes, CTCs, circulating tumor

DNA (ctDNA), proteins, and exosomes. Following separation, the captured cells and

biomolecules can either be extracted for downstream genomic/proteomic analysis (e.g.,

single-cell RNA sequencing or digital PCR) or directly analyzed on-chip through

integrated detection modules (e.g., impedance cytometry for cellular characterization

or surface-enhanced Raman spectroscopy (SERS) for protein profiling). This "sample-

in-answer-out" capability positions microfluidic systems as transformative tools for

liquid biopsy applications, offering advantages in automation, multiplexing, and

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