Microfluidic Liquid Biopsy Minimally Invasive Cancer Diagnosis by Nano-Plasmonic Label-Free Detection of Extracellular Vesicles: Review
Abstract
1. Introduction
1.1. Classes of Analyte in Liquid Biopsy
1.1.1. Circulating Tumor Cells (CTC)
1.1.2. Circulating Cell-Free Nucleic Acids (Cf-NAs)
1.1.3. Tumor-Educated Platelets (TEPs)
1.1.4. Exosomes
1.2. Hurdles in Adapting Exosomes in Liquid Biopsy
1.3. Handling and Analysis of Exosomes: A Microfluidic Approach
2. Extracellular Vesicles: Exosomes, Microvesicles, and Others
2.1. Exosomes
2.2. Microvesicles
Exosomes | Microvesicles | Apoptotic Bodies | |
---|---|---|---|
Size | 20–100 nm | 50–1000 nm | 500–2000 nm |
Density | 1.13–1.19 g/mL | 1.04–1.07 g/mL | 1.16–1.28 g/mL |
Biogenesis | Formed through inward luminal budding of the membrane and fusion of multivesicular bodies with the cell membrane (endolysosomal pathway). | Directly shed from the cell’s plasma membrane through outward budding. | Formed through membrane blebbing from a cell undergoing apoptosis. |
Composition |
|
|
|
Biomarker | Tetraspanins family (such as TSPAN29 and TSPAN30, CD81, CD82, CD9, CD63), ESCRT proteins (Alix, TSG101), actin, flotillin, Hsc70, HSP 90, Hsp60 and Hsp20 clathrin, integrins (such as α3, α4, β1, β2) | Integrins, selectins, flotillin-2, CD40 ligand, metalloproteinase | Annexin V positivity, phosphatidyl serine |
2.3. Apoptotic Bodies
3. Microfluidic Platform for EV Isolation, Detection, and Characterization
3.1. EV Isolation
3.1.1. Physical Technique for EV Isolation in a Microfluidic Platform
3.1.2. Asymmetric Flow Field-Flow Fractionation (A4F)
3.1.3. Electrical Technique for EV Isolation in Microfluidic Platform
3.1.4. Immunoaffinity-Based Techniques for EV Isolation in Microfluidic Platform
3.2. EV Detection
4. Plasmonic Technologies for Exosome Analysis
4.1. Raman Scattering
4.2. Surface-Enhanced Raman Scattering
4.2.1. Physical Modification of SERS Substrate
4.2.2. Chemical Modification of SERS Substrate
4.2.3. SERS Signal Detection Analysis
4.3. Surface Plasmon Resonance
4.4. Surface Plasmon Resonance Imaging
4.5. Localized Surface Plasmon Resonance (LSPR)
5. Research Outlook and Conclusions
Author Contributions
Funding
Conflicts of Interest
References
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Tests (Company) ☐SC|☐D|☐PT | Cancers | Marker | Sample | Comments | Refs. |
---|---|---|---|---|---|
Guardant360 (Guardant Health, Palo Alto, CA, USA) ☑D | Lung (NSCLC), Breast, Colorectal, Prostate | 100 + ctDNA | Blood | Effective for advanced stage (stage III or IV) cancer. FDA approved in 2020. | [8,9,10] |
FoundationOne Liquid (Foundation Medicine, Boston, MA, USA) ☑D | NSCLC | 70 + ctDNA | Blood | For patients with advanced cancer. FDA approved in 2020. | [11] |
Epi pro Colon (Epigenomics AG Heidelberg, Germany) ☑SC, ☑D | colorectal cancer | methylated Septin 9 DNA | Blood | For individuals aged 50 and older who are at average risk for colorectal cancer, this is the first blood-based test available. FDA approved in 2016. | [12] |
Cologuard (Exact Sciences, Madison, WI, USA) ☑D | colorectal cancer | DNA | Stool | The first non-invasive DNA screening test for colorectal cancer is intended to screen adults 45 years of age and older who are at average risk for colorectal cancer. FDA approved in 2014. | [13] |
CELLSEARCH CTC kit (Menarini Silicon Biosystems, Inc., Huntingdon Valley, PA, USA) ☑D | Breast, Prostate and Colorectal | CTC | Blood | Test approved by Health Canada in 2010. First FDA-cleared test (2004) for the enumeration of circulating tumor cells in peripheral blood. | [14] |
Laboratory-developed test (LDT) regulated by Clinical Laboratory Improvement Amendments (CLIA) | |||||
Galleri (GRAIL, Menlo Park, CA, USA) ☑SC | Multiple cancer screening | ctDNA (methylation markers and machine learning for cancer detection) | Blood | [15] | |
OncoBEAM (Sysmex Inostics, Baltimore, MD, USA) ☑SC, ☑PT | Lung, Colorectal, and other | ctDNA | Blood | [16,17] | |
CancerSEEK (Johns Hopkins University, Baltimore, MD, USA) ☑SC | Ovarian, Liver, Stomach, and other. | ctDNA | Blood | ctDNA and protein markers for early cancer detection. | [18] |
Pathfinder (Freenome, Brisbane, CA, USA) ☑SC | Colorectal cancer | ctDNA | Blood | a combination of DNA, RNA, and protein biomarkers. | [19] |
Cancer Type | Exosomal Biomarker | Biomarker Type | Biofluid | Indication | Clinical Sample Size | Refs. |
---|---|---|---|---|---|---|
Lung cancer | miR-222-3p | miRNA | Serum | Prognosis | TP N = 50 | [56] |
EGFR T790M | mRNA | Plasma | TP N = 84 [57] | [57,58] | ||
miR-181-5p, miR-30a-3p, miR- Adenocarcinoma-specific: 30e-3p, and miR-361-5p were SCC specific: miR-10b-5p, miR-15b-5p, and miR-320b | miRNA | Plasma | TP N 46, HC N = 42, S = 60 | [59] | ||
miR-193a-3p, miR-210-3p and miR-5100 | miRNA | Bone marrow and Plasma | Diagnosis and prognosis | TP N = 41, HC N = 30 | [60] | |
circSATB2 | Circular RNA | Cell line H460, A549 and H1299 | [61] | |||
NY-ESO-1 | Antigen | Plasma | [62] | |||
TTF-1 and miR-21 | Protein | Serum | Diagnosis | NA | [63] | |
Breast cancer | miR-1246 | miRNA | Plasma | Diagnosis | TP N = 46, HC N = 28 | [64] |
miR-21, miR-105 and miR-222 | miRNA | Serum | Diagnosis | TP N = 53, HC N = 8 | [65] | |
Glycoprotein (MUC1) | Protein | Cell line MCF7&MDA-MB-231 | [66] | |||
PKG1, RALGAPA2, NFX1, TJP2 | Protein | Plasma | [67] | |||
HER2 | Protein | Plasma | [68] | |||
CD82 | Protein | Serum and plasma | Diagnosis | TP N = 80, BTP N = 80, HC N = 80 | [69] | |
miR-375 | miRNA | Serum | Diagnosis | TP N = 17, HC N = 12 | [70] | |
Gastric cancer | circSHKBP1 | Circular RNA | Serum | Diagnosis | TP N = 20, HC N = 20 | [71] |
HOTTIP | long non-coding RNA | Serum | Diagnosis and prognosis | TP N = 126, HC N = 120 | [72] | |
Early-stage GC | lncUEGC1 | long non-coding RNA | Plasma | Diagnosis | TP N = 10, HC N = 5 | [73] |
Rectal cancer | miR-30d-5p, miR-181a-5p and miR-486-5p | miRNA | Plasma | Diagnosis and prognosis | TP N = 24, HC N = 5 | [74] |
HCC | tRNA-ValTAC-3, tRNAGlyTCC-5, tRNA-ValAAC-5, and tRNA-GluCTC-5 | miRNA | Plasma | Diagnosis | TP N = 35, HC N = 11 | [75] |
circUHRF1 | Circular RNA | Plasma | Diagnosis | TP N = 240, HC N = 20 | [76] | |
Early-stage HCC | miR-21 and miR-10b | miRNA | Serum | Prognosis | TP N = 124 | [77] |
Pancreatic cancer | KRAS | mRNA | Plasma | Diagnosis and prognosis | TP N = 127, HC N = 136 | [78,79] |
CKAP4 | Protein | Serum | Diagnosis | TP N = 47, HC N = 18 | [80] | |
Glypican 1(GPC1), Migration inhibition factor (MIF) | Protein | Serum | TP N = 71, HC N = 32 | [81] | ||
Prostate cancer | AR-V7 | Androgen receptor | Plasma | Prognosis | TP N = 36 | [82] |
miR-196a-5p and miR-501-3p | miRNA | Urine | Diagnosis | TP N = 48, HC N = 28 | [83] | |
miR-1290 and miR-375 | miRNA | Plasma | TP N = 23, HC N = 50 | [84] | ||
PSA | Antigen | Plasma | [85] | |||
GGT1 | Gene | Serum | [86] | |||
PTENP1 | Gene | Plasma | Diagnosis | TP N = 50, HC N = 60 | [87] | |
Bladder cancer | lncLNMAT2 | long non-coding RNA | Serum and urine | Diagnosis and prognosis | TP N = 206, HC N = 120 | [88] |
lncRNAs(SPRY4-IT1, MALAT1 and PCAT-1) | long non-coding RNA | Urine | Diagnosis and Prognosis | TP N = 184, HC N = 184 | [89] | |
Colorectal | Glypican-1(miR-96-5p and miR-149) | miRNA | Plasma | [90] | ||
CEA | Antigen | Serum | [91] | |||
Cholangiocarcinoma | AMPN, VNN1, PIGR | Gene | Serum | [92] | ||
Ovarian cancer | E-cadherin | Protein | Ascites | Diagnosis and prognosis | TP N = 35, HC N = 6 | [93] |
miR-200b and miR-200c | miRNA | Serum | Diagnosis and prognosis | TP N = 163, BTP N = 20, HC N = 32 | [94] | |
CD24, EpCAM, CA-125 | Protein | Plasma | [95] | |||
Cervical cancer | let-7d-3p and miR-30d-5p | miRNA | Plasma | Diagnosis | NA | [96] |
Multiple Melanoma | Ig-BCR | Cell receptor | Serum | Diagnosis | Serum of 5T33MM engrafted mice | [97] |
let-7b and miR-18a | miRNA | Serum | Prognosis | TP N = 156, HC N = 5 | [98] | |
PMSA3 and lncPMSA3-AS1 | Gene | Serum | Prognosis | Bortezomib resistance N = 12, bortezomib sensitivity N = 45 | [99] | |
Melanoma | PD-L1 | Protein | Plasma | Diagnosis and prognosis | TP N = 44, HC N = 11 | [100] |
BRAFV600E | Gene | Plasma | Prognosis | TP N = 12, HC N = 12 | [101] | |
Glioblastoma | EGFR vIII | mRNA | Serum | Diagnostic | [102] |
Optical Methods | Non-Optical Methods |
---|---|
Optical microscopy | Scanning electron microscope (SEM) |
Dynamic Light Scattering (DLS) | Transmission Electron Microscopy (TEM) |
Nano Tracking Analysis (NTA) | Atomic force microscopy (AFM) |
Fluorescence microscopy | Impedance flow cytometry |
Surface Plasmon Resonance (SPR) | Tunable Resistance Pulse Sensing (TRPS) |
Localized Surface Plasmon Resonance (LSPR) | Mass spectroscopy |
Biomarker | Cancer Type (Cell Line) | Sample | Detection Technique (Target Molecule) | Statistics | Refs. |
---|---|---|---|---|---|
CD63 | Glioma (U251) | Cell culture | SPR (Antibody) | 4.23 × 10−3 μg/mL | [156] |
CD24 EpCAM | Ovarian (CaOV3) | Cell culture | SPR (Antibody) | 1000+ sites for inspection | [143] |
EGFR PD-L1 | Non-small cell lung cancer (A549) | Cell culture, Serum | SPR (Antibody) | sensitivity of 9.258 × 103%/RIU and resolution of 8.311 × 10−6 RIU. | [146] |
HER2 | Breast cancer (BT474) | Cell culture, Serum | (SPR) (Antibody) | LOD: 2070 Exosomes/μL | [145] |
HSPG | Multiple myeloma | Serum | SPR (Protein) | LOD: 5 ng/mL | [142] |
HSP90 | Breast cancer (MCF7) | Cell culture | LSPR (Vn96) | No data | [155] |
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Neriya Hegade, K.P.; Bhat, R.B.; Packirisamy, M. Microfluidic Liquid Biopsy Minimally Invasive Cancer Diagnosis by Nano-Plasmonic Label-Free Detection of Extracellular Vesicles: Review. Int. J. Mol. Sci. 2025, 26, 6352. https://doi.org/10.3390/ijms26136352
Neriya Hegade KP, Bhat RB, Packirisamy M. Microfluidic Liquid Biopsy Minimally Invasive Cancer Diagnosis by Nano-Plasmonic Label-Free Detection of Extracellular Vesicles: Review. International Journal of Molecular Sciences. 2025; 26(13):6352. https://doi.org/10.3390/ijms26136352
Chicago/Turabian StyleNeriya Hegade, Keshava Praveena, Rama B. Bhat, and Muthukumaran Packirisamy. 2025. "Microfluidic Liquid Biopsy Minimally Invasive Cancer Diagnosis by Nano-Plasmonic Label-Free Detection of Extracellular Vesicles: Review" International Journal of Molecular Sciences 26, no. 13: 6352. https://doi.org/10.3390/ijms26136352
APA StyleNeriya Hegade, K. P., Bhat, R. B., & Packirisamy, M. (2025). Microfluidic Liquid Biopsy Minimally Invasive Cancer Diagnosis by Nano-Plasmonic Label-Free Detection of Extracellular Vesicles: Review. International Journal of Molecular Sciences, 26(13), 6352. https://doi.org/10.3390/ijms26136352