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surpass - traditional analytical methods like ELISA, PCR, and mass spectrometry.

Particularly in scenarios requiring portability, automation, or multiplexed detection,

microfluidic platforms are increasingly becoming the preferred choice, suggesting they
are ready to transition from research laboratories to widespread clinical and industrial

adoption. Microfluidic models for molecular diagnostics have emerged as powerful

platforms for comprehensive biomarker analysis, particularly through innovative

device designs enabling back-to-back processing of circulating tumor cells (CTCs),

cell-free DNA (cfDNA), tumor-derived exosomes, and protein biomarker isolation

from minimally processed whole blood (Figure 7A) 127 .

(1) Circulating Nucleic Acid Separation

Circulating cell-free DNA (cfDNA), primarily released by apoptotic or necrotic

cells, includes tumor-derived mutant fragments termed ctDNA 128,129 . CtDNA is a

highly tumor-specific biomarker (Figure 7B) 130–132 . However, elevated cfDNA levels

also occur in benign conditions or tissue injury, overlapping with early-stage cancer

concentrations 133 . Detection systems must therefore discriminate and quantify mutant

allele frequencies 134 . Compared to CTC isolation, integrated microfluidic devices for
direct quantitative cfDNA isolation from whole blood remain scarce, largely due to

extraction challenges from minute volumes and complex preprocessing. Recent

advances include nanostructured microelectrode electrochemical sensors enabling

amplification-free, direct detection of mutant ctDNA in serum 135–137 .

(2) Tumor-Derived Exosome Enrichment

Exosomes are extracellular vesicles actively secreted by cells, including tumor

138
cells . They carry molecular cargo (DNA, RNA, proteins, lipids) harboring rich tumor
information. Highly abundant in body fluids and more stable than ctDNA, exosomes

enable detection in early-stage cancers, making them promising biomarkers (Figure

7C) 139 . Traditional isolation (ultracentrifugation, precipitation, filtration) is laborious,

time-consuming, and yields suboptimal purity. Microfluidic techniques primarily use

immunoaffinity capture. The EVHB chip, with a Y-configuration, processes milliliters

of serum; its nanostructured surface captures vesicles across size ranges, outperforming

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