Harnessing the Potential of Nanotechnology for Liquid Biopsy of Cancer

Abstract

Liquid biopsy offers dynamic and noninvasive analysis of cellular biomarkers, thereby presenting enormous potential for early detection of cancer, cancer staging, prediction of relapse, real-time examination of therapeutic efficacy, perception of therapeutic targets, and understanding the resistance mechanisms. Nanotechnology has emerged as a novel tool to widen the application horizon of liquid biopsy. Several nanomaterials, nanodevices, nanostructures, and nanosensors have been explored for improved application of liquid biopsy for biomarker detection. The circulating tumor cells (CTCs), circulating tumor proteins (CTP), miRNA and extracellular vesicles (EVs) are some of the important biomarkers for detection by liquid biopsy in bodily fluids. Herein, we have discussed the state of the art and beyond in advances in nanotechnology and in increasing the specificity, sensitivity, and purity with which we detect liquid biopsy biomarkers. The opportunities and prospects of these advanced innovative nanomaterials and technologies in clinical applications are explored. Furthermore, various isolation and biosensing strategies for visualization and signal amplification using nanomaterials are summarized. The utilization of nanotechnology-based liquid biopsy may provide greater insights for improved treatment, diagnosis, and prognosis of cancer.

1. Introduction: Crystal Clear Nano and Liquid Biopsy

Cancer is one of the most devastating diseases and a notable cause of mortality across the globe, and the numbers of cases are increasing each year at an alarming pace. Until now, the majority of cancer patients have experienced poor prognosis, especially when diagnosed in an advanced stage with metastasis [1,2]. One of the chief reasons for rapidly increasing death is failure in diagnosing at an early stage. So, to resolve this issue, detection at earlier stages is highly desirable. In this context, liquid biopsy provides a unique platform for early detection of cancer without any invasive procedures or surgical intervention [3,4,5,6]. “Liquid biopsy” is a minimally invasive method for finding biomarkers of cancer in bodily fluids, most commonly blood but also urine, saliva, and cerebrospinal fluid. Although hematologic malignancies are also being investigated, liquid biopsy is applicable to a broad spectrum of cancers, including solid tumors like breast, lung, and colorectal cancer. Liquid biopsy deals with analysis or detection of genetic abnormalities (mutations, epigenetic modifications, etc.) and exploration of biomarkers such as circulating tumor cells (CTCs), cell-free nucleic acids (cfNAs), circulating tumor proteins (CTPs), miRNA, and extracellular vesicles (EVs) in bodily fluids, specifically the peripheral blood (Figure 1) [7]. In cases of metastasis, CTCs escape from the original site and enter the bloodstream. These cells move along with the circulation and subsequently arrive at a secondary site, forming a tumor colony. CTCs are found to be linked with tumor initiation and metastasis and have shown excellent prognostic and diagnostic potential [8,9]. Additionally, cell-free nucleic acids (cfNAs) are present in human blood, and it has been found that alterations in cfNAs can lead to cancer [10,11]. Small-noncoding RNAs, i.e., microRNAs (miRNAs), regulate the translation of protein-coding genes, and it has been found that those alterations or modifications in miRNA expression are associated with cancer progression. Furthermore, endosome-derived extracellular vesicles (EVs) play an important role in antigen presentation and in immune response. These EVs are generally secreted by all the mammalian cells and are abundantly present in bodily fluids including the blood. They are involved in various physiological processes; they function as communicating agents by transferring several biomolecules such as lipids, proteins, and nucleic acids (mRNA and microRNAs (miRNA)) from their origin to target cells; and they activate tumor progression and metastasis. They are thus considered an emerging liquid biopsy tool [12,13,14]. Liquid biopsy holds immense potential for early noninvasive diagnosis, prognosis, disease progression monitoring, assessment of treatment effectiveness, and detection of therapeutic targets for development of effective drug targeting [15]. Various techniques and methodologies have been utilized for the detection and analysis of these liquid biopsies, and the importance of liquid biopsies in the field of cancer has been demonstrated. Nanotechnology has made significant progress in further improving the potential of liquid biopsy. Advancements in nanomaterials can aid in the improvement of specificity, sensitivity, and selective detection of CTCs, cfNAs, TEPs, and EVs [7,16,17].

Nanotechnology—which is distinguished by the use of structures, devices, and materials whose size ranges from 1 nm to 1000 nm—possesses potential advantages in liquid biopsy like prominent sensitivity, simplicity, rapid detectability, analysis, lower cost, and the possibility for portability and personalized medicine [18,19,20,21]. The present review discusses the role of nanotechnology in liquid biopsy applications. This narrative review is based on works published between 2013 and 2024 that were retrieved from PubMed, Web of Science, and Google Scholar. We focused on English-language studies that examined tools based on nanotechnology for liquid biopsy applications in cancer. The search terms “nanotechnology,” “liquid biopsy,” “cancer diagnostics,” “extracellular vesicles,” “ctDNA,” “ctRNA,” “nanoparticles,” and “biosensors” were used for this review. Studies involving recent technology advancements, which showed preclinical or clinical relevance, or provided sensitivity/specificity measurements were preferred. Only when important to provide historical background and when referring to foundational publications were studies more than ten years old included.

2. Using Nanotechnology to Aid the Clinical Translation of Liquid Biopsy-Based Diagnostics

Nanotechnology offers a variety of advantages because of their nanodimensional structures which display enhanced optical, electrical, mechanical, magnetic and thermal features to generate empirical signals for diagnosis at an earlier stage and for concurrent monitoring during the treatment phase [22,23]. It provides highly sensitive and efficient detection of liquid biopsy biomarkers, as their concentration is very low in biological fluids. To overcome these limitations, nanotechnology has been used to increase the surface-area-to-volume ratio that in turn facilitates enhanced association of nanomaterials and biochemical analytes to increase the capturing of targeted cells in the blood associated with cancer [24]. Moreover, the extremely tiny dimensions of nanostructures promote the capturing and enrichment of nanoscale biomarkers like EVs (30–100 nm) and facilitate the amplification of localized signals from EVs [18]. Detection of CTCs, EVs, and cfNAs is very difficult in the bloodstream, as the bloodstream contains normal blood cells, cellular debris, and many other things that shield the signals originating from the CTCs and EVs, thereby impeding their capturing. In this regard, nanomaterials and nanostructures offer a unique platform for precisely detecting liquid biopsy biomarkers [18]. Additionally, cancers involve different cell populations, which are closely associated with cancer progression and drug resistance [25]. The detailed roles of CTCs and EVs are not fully known yet. However, in this context, nanotechnology-based approaches provide versatile and integrated platforms such as the lab-on-chip detection and further downstream molecular analysis of liquid biopsy biomarkers such as EVs and CTCs for future drug discovery, which hold enormous potential for clinical translation. The safety profile and biocompatibility of nanostructures not only enable the efficient capture of CTCs and EVs but also preserve the integrity of cellular biomarkers, which is crucial for in vitro and downstream molecular characterization [26]. Several nanomaterials are being developed and applied in clinical settings that strengthen the potential of nano-based approaches for improving the sensitivity of liquid biopsy for early detection, treatment response monitoring, disease prediction, and point-of-care testing [27,28,29]. Nanomaterials like nanorods, nanofibers, nanowires, and nanoparticles have been synthesized to increase the capacity to capture cancer-related abnormalities in fluids. By virtue of their nanoscale size, nanostructures exhibit amplified responses and increased surface area for interactions between materials and biomolecules, resulting in enhanced specificity and binding efficiency, as shown in

Table 1. Utility of nanotechnology for the isolation of CTCs and EVs.

S. No	Detection Tool	Diagnostic Marker	Clinical Applications	Advantages	Ref
CTC Isolation
1.	Peptide-modified magnetic nanoparticles (MNPs)	EpCAM and HER2	-	Low cost, constant properties and maintenance of the CTCs in a viable state	[30,31]
2.	Magnetic nanospheres	EpCAM	Utilized in lung, liver, colon, and breast cancer blood sample	Rapid, sensitive, greater reliability and reproducibility, maintains the viability of the isolated cells	[32]
3.	Cell membrane-coated magnetic nanoparticles	EpCAM	-	Biocompatible in nature, eliminates the risk of non-specific binding of WBCs, great capture efficiency of CTCs	[33]
5.	CellSearch (MNPs)	EpCAM	-	Good reliability and can be employed for routine examination of cancer patients	[34,35]
6.	GO nanosheets	EpCAM	Early-stage lung cancer patients, breast, and pancreatic cancer blood sample	Enhanced capture potential, specificity, and sensitivity	[36,37]
7.	Silicon nanowires	EpCAM and DNA in cells	-	Efficient and promotes specified targeting and release of CTCs	[38,39]
8.	Polymer-grafted silicon nanowires	EpCAM	-	Enhanced capture efficiency	[40]
9.	Silicon nanowires	Sialic acid	-	Efficient cell capture and release	[41]
10.	Polysaccharide nanofiber-embedded chip	EpCAM and CD146	Two stage-IV melanoma patients blood sample	Isolation and sequencing of CTC	[42,43]
11.	TiO2 nanofibers	EpCAM	Colorectal cancer and gastric cancer patient-derived blood samples	High affinity	[44]
13.	Nanopillars	EpCAM	-	Convenient, Efficient, and cost-effective	[45,46]
14.	Nanodots	EpCAM	-	Efficient	[46]
EV Isolation
15.	GO-modified nanostructure	CD81	-	Excellent isolation efficiency and minimal non-specific adsorption	[47]
16.	Nano-DLD silicon arrays	-	-	Fast and sensitive, label-free, small sample requirement	[48]
17.	Nanoporous isolation chip (Nanostructured surface)	-	Highly efficient plasma ultracentrifugation of lung cancer patients	Inexpensive, simple, fast, good-yield, and easy handling	[49]

.

In recent times, various metallic and non-metallic nanostructures have been fabricated and demonstrated for enhanced CTC, EV, cfNA, etc. capture and detection. Magnetic nanoparticles (MNPs) are the classically used nanomaterials for detecting CTCs. Due to their high surface-to-volume ratio, rapid binding kinetics, and stability in solution, nanostructures offer high binding capacity, efficient target capture, and rapid enrichment, making them ideal for use in liquid biopsy to detect CTCs and related components [31,50,51]. The CellSearch system is clinically approved for the detection of circulating tumor cells (CTCs) in patients with breast and colorectal cancers. This system utilizes magnetic ferrofluids for the detection and capture of CTCs from blood samples. It is based on automated immunomagnetic enrichment of epithelial tumor cells that express the epithelial cell adhesion molecule (EpCAM). Specifically, magnetic ferrofluids conjugated with anti-EpCAM antibodies are added to 7.5 mL of peripheral blood collected from the patient, enabling selective isolation of EpCAM-positive CTCs [52,53]. Subsequently, the enriched sample is stained for nuclear staining by immunocytochemistry for cells expressing cytokeratin 8/18/19, whereas pan-leukocyte marker CD45 is utilized for eliminating contaminating leukocytes. Finally, based on consensus guidelines, a trained user using an automated fluorescence microscope captures and quantifies all the cells that meet the criteria for CTCs accordingly. The CellSearch system is highly specific and very sensitive, as it can capture even a single CTC in 7.5 mL of blood with minimal inter-reader variability [54,55,56]. A schematic of the CellSearch system is shown in Figure 2. Recently, Peng et al. and Bai et al. used the HER2 and EpCAM recognition peptide conjugated with MNPs to capture the CTCs expressing these two biomarkers on their surface. These conjugated MNPs demonstrated high capture efficiency and high sensitivity for CTC detection in breast, liver, and prostate cancers, achieving a capturing rate of over 90% for CTCs and 68% for HER2-expressing CTCs and significantly outperforming antibody-functionalized MNPs [30,31].

Gold nanoparticles are extensively employed for detection in biological systems and display great sensitivity and specificity at very low concentrations [57]. They have enhanced cell adhesion properties in their nano-form because of their increased surface interface. Furthermore, when they are functionalized with antibodies with different biological domains like peptides, antibodies, aptamers, etc., they show enhanced detection efficiency with an increased capture rate. Hyeun et al. tested the capture efficiency and sensitivity of functionalized graphene oxide (GO) nanosheets on a patterned gold surface for CTCs in breast, lung, and pancreatic cancer patients’ blood. Using the principle of electrostatic attraction, nanosheets of GO were adsorbed on the patterned gold surface followed by functionalization with biotinylated EpCAM antibodies. This fabricated material was examined to capture CTCs and was found to be successful for the detection of CTCs even at low concentrations of CTCs in patient-derived blood with promising sensitivity (3 ± 32.4% at 3–5 cells/mL blood) [36]. Furthermore, gold nanoparticle-based biosensors and microfluidic devices have been developed and have attracted interest in recent years [58]. The fabricated devices were able to detect the different biomolecular markers for different diseases from diverse array of samples that include serum, whole blood, saliva, and urine.

Recently, gold nanoparticle-based graphene quantum dots conjugated with the neuron-specific enolase (NSE) antibodies showed remarkable diagnosis of small cell lung cancer with as low as 0.09 pg/mL of NSE in blood [59]. Surface-enhanced Raman scattering (SERS) is widely used for the highly sensitive detection of multiplex biological components, offering molecular specificity, single-molecule sensitivity, and resistance to quenching. Leveraging these advantages, various metallic nanostructures with well-controlled sizes and shapes have been developed [60]. For direct detection of CTCs without a pre-purification step from the patient’s blood, Wu et al. designed gold-based SERS nanoparticles. They used the Raman reporter molecule 4-mercaptobenzoic acid (4-MBA) and modified the nanoparticles to attain strong SERS signals. For specific CTC detection, they conjugated the nanoparticles with folic acid (FA) and stabilized the nanoparticles with bovine serum albumin (BSA) to avoid any non-specified cell binding. The authors synthesized gold nanoparticles of various shapes and found that gold nanostars exhibited the highest sensitivity, achieving a detection limit of 1 cell/mL of blood, which is significantly lower than the previously reported limit of 5 cells/mL [61].

Similarly, graphene oxide (GO)-based materials were also assessed in various studies to examine their potential in improving liquid biopsy-based capturing of cancer biomarkers. In this regard, Zhang et al. proved the tremendous potential of GO to enhance sensitivity as well detection efficiency. They fabricated a nano-interfaced microfluidic EV device developed by coating a layer-by-layer channel and Y-shaped micro posts with antibody-conjugated GO-induced nanostructured polydopamine. This system captured the EVs with a lowest limit of 50/μL with a 4-log dynamic range. For clinical translational purposes, they used this device to differentiate ovarian cancer patients from healthy blood donor individuals, and they were successfully able to identify the cancer-bearing patients [47]. As a cell affinity substrate, NanoVelcro offers an exclusive method of capturing CTCs. In this system, compactly arranged and vertically oriented silicon nanopillars (SiNPs) are used and conjugated with specific antibodies, which results in enhanced capture of CTCs. SiNPs improve the interactions or binding kinetics between cells and substrates, which remarkably increases the capture efficiency in comparison with unstructured substrates [45,46,62]. Similarly, TiO₂ nanofibers (TiNFs) were synthesized via electrospinning and subsequently functionalized with antibodies for CTC capture. The horizontally aligned electrospun TiNFs provided enhanced interaction and binding affinity with cells and cellular components, thereby improving detection sensitivity and capture efficiency. Using this approach, TiNFs substrates conjugated with anti-EpCAM were utilized to detect the CTCs in blood samples of three colorectal and seven gastric cancer patients. Of the three colorectal cancer patients, in two patients, 0–2 CTCs/0.5 mL of blood sample was detected, and in all seven gastric cancer patients, CTCs in the range of 3–19 were detected per 0.5 mL of blood sample. This highly efficient and sensitive approach indicated the potential of nanotechnology in improving liquid biopsy-based cancer diagnosis especially at early stages when only a few CTCs are roaming around in the patient’s blood [44].

Some in vitro studies have demonstrated that DNA catalytic reactions can detect nucleic acids with a high degree of sensitivity [63,64,65]. Inspired by those findings, Hu et al. fabricated a lipid–polymer hybrid nanoparticle (LPHN)-catalyzed hairpin DNA circuit (CHDC) biochip which offers very sensitive quantification and imaging of target RNAs in EVs. A specific CHDC containing two hairpin DNAs and a reporter for glypican-1 (GPC1) mRNA was encased in LPHNs and then fixed on a thin glass slide. These cationic charged LPHNs were capable to target negatively charged EVs by virtue of electrostatic interactions followed by formation of a used LPHN–EV nanocomplex, resulting in the mixing of CHDC with target RNA. CHDC is prompted by hybridizing with target RNA and rapidly generates amplified signals. The ultrahigh sensitivity of the fabricated LPHN–CHDC fusion complexes was evaluated by targeting GPC1 mRNA in EVs from pancreatic cancer. This design was capable of capturing EVs at concentrations as low as 10⁵/mL, which is equivalent to approximately 60 EVs/μL. Furthermore, LPHN-CHDC also distinguished the early-to-late-stage PDAC patients from healthy donors and other patients with benign pancreatic disorders [66]. Moreover, apart from the discussed applications, there are several ongoing clinical trials for liquid biopsy which are tabulated in

Table 2. Ongoing clinical trials for application of liquid biopsy for cancer diagnosis.

S. No	Study Objective/Title	Sample Size	Intervention	Status/Study Type	Disease Condition	Trial ID
1.	Ovarian cancer detection by TEPs and ctDNA	500	TEP, ctDNA	Observational	Ovarian neoplasms	NCT04022863
2.	Ovarium Cancer Detection by TEPs and ctDNA	100	ctDNA and CTCs	Interventional	Invasive breast carcinoma	NCT04223492
3.	Treatment of high-risk stage II and stage III colon cancer patients post-surgery using liquid biopsy: the PEGASUS trial	140	ctDNA	Phase 2/Interventional	Colon cancer	NCT04259944
4.	Assessment of early detection based on liquid biopsy in lung cancer (ASCEND-LUNG)	467	cfDNA methylation	Observational	Lung cancer	NCT04817046
5.	Detecting resistance against targeted therapy in prostate cancer patients by liquid biopsy (PEARL)	120	CTCs	Observational	Prostate cancer	NCT03601143
6.	Advance detection of stage IV relapses in colorectal cancer patients (REDCLOUD)	141	cfDNA	Observational	Stage IV colorectal cancer	NCT04232891
7.	Liquid biopsy evaluation and repository development at princess Margaret (LIBERATE)	2500	cfNA, cfDNA, cfRNA	Observational	Solid tumor or hematological malignancy	NCT03702309
8.	NSCLC heterogeneity in early-stage cancer patients and predicting relapse through personalized liquid biopsy	50	ctDNA and CTCs	Interventional	Non-small cell lung carcinoma	NCT03771404
9.	Sample collection from patients with non-hematologic cancer and use in developing a liquid biopsy assay	3000	(ctDNA)	Observational	Non-hematologic cancer	NCT02586389
10.	Clinical Application of Liquid Biopsy for Precise Diagnosis and Prognosis in Lymphoma	50	cfDNA	Observational	Lymphoma	NCT04062877
11.	Multicenter liquid biopsy for verifying assumed biomarkers for the treatment of pancreatic cancer	662	ctDNA	Observational	Pancreatic adenocarcinoma	NCT04241367
12.	Early detection of prostate cancer (PROLIPSY)	320	CTCs, cfDNA, exosomes	Interventional	Prostate cancer	NCT04556916
13.	Liquid biopsy in mature B-cell tumors	444	cfDNA,	Observational	Mature B-Cell neoplasm	NCT03280394
14.	Pan-cancer early-stage detection by liquid Biopsy technique project (PRESCIENT)	11879	cfDNA,	Observational	Cancer	NCT04822792
15.	Preventing viral pandemic-associated risk of cancer death using less invasive diagnostic tests—liquid biopsies (PREVAIL ctDNA)	112	ctDNA	Observational	Colorectal, lung, bladder, pancreatic biliary tract neoplasm	NCT04566614
16.	Therapeutic resistance and clonal evolution assessed with liquid biopsy in ICI-treated NSCLC Patients	250	ctDNA	Observational	Lung cancer	NCT04566432
17.	Liquid biopsy in analysing the Operation in Gastric Cancer and Neoadjuvant Chemotherapy	40	CTC, ctDNA, cfDNA	Phase 2 Interventional	Gastric cancer	NCT03957564
18.	Cambridge liquid biopsy and tumour profiling study for patients on experimental therapeutics trials (CALIBRATE)	100	ctDNA	Observational	Cancer	NCT02994511
19.	The tracking molecular evolution for NSCLC (T-MENC) study	200	ctDNA	Observational	Lung cancer	NCT03838588
20.	Circulating DNA to improve outcomes of oncology patients—a randomized study (COPE)	332	ctDNA	Phase 2 interventional	Colorectal cancer and non-small cell lung cancer	NCT04258137
21.	Detecting MSI in circulating tumor DNA of colorectal carcinoma patients	35	cfDNA	Observational	Colorectal cancer stage IV	NCT03594448
22.	Prognostic role of circulating tumor DNA in resectable pancreatic cancer (PROJECTION)	200	ctDNA	Observational	Pancreatic cancer	NCT04246203
23.	Liquid biopsy using methylation sequencing for lung cancer	280	ctDNA	Observational	Lung cancer	NCT04253509
24.	Advanced Analysis of Single Cells in Colorectal Cancer	45	CTCs	Observational	Metastatic liver carcinoma and stage IV colorectal cancer	NCT02809716
25.	Phenotypic spectrum of CTCs in tumors of the female reproductive system (CTCs)	150	CTCs	Observational	Breast cancer, ovarian cancer, endometrial cancer	NCT04817501
26.	Fluid biopsy for the diagnosis of lung cancer	75	ctDNA	Observational	Lung carcinoma	NCT04162678
27.	Evaluation of cfDNA as a marker of response in rectal cancer	120	cfDNA	Interventional	Cancer of rectum	NCT04319354
28.	Lung cancer biomarkers for risk stratification	590	ct-DNA LUNAR assay	Observational	Non-small cell lung cancer	NCT03774758
29.	Study of circulating tumor DNA (ctDNA) kinetics in immuno-oncology (IO-KIN)	20	ctDNA	Observational	Head and neck cancer, advanced metastatic cancer and squamous cell carcinoma	NCT04606940
30.	A study on differentiation of benign and malignant nodules and noninvasive advanced diagnosis of stromal and gastrointestinal tumors	300	cfDNA	Observational	Gastrointestinal stromal tumor	NCT04143048
31.	Monitoring of serial circulating tumor DNA (ctDNA) during adjuvant capecitabine in early triple-negative breast cancer	25	ctDNA	Interventional	Breast cancer	NCT04768426
32.	ctDNA as a biomarker for treatment response in HNSCC (PECAN)	70	ctDNA	Interventional	Squamous cell carcinoma of head and neck	NCT03540563
33.	Analysis of cfDNA in patients with hepatocarcinoma and treated by sorafenib or regorafenib (HELP)	70	cfDNA	Interventional	Hepatocarcinoma	NCT03956940
34.	Active surveillance in early lung cancer (ACTION-LUNG)	600	ctDNA	Observational	Colorectal cancer pancreatic cancer biliary tract cancer	NCT04776837
35.	Monitoring efficacy of radiotherapy in lung cancer and esophageal cancer	26	ctDNA CTCs	Interventional	Lung cancer	NCT03923777
36.	Mechanisms of resistance in EGFR mutated non-pretreated advanced lung cancer receiving osimertib (MELROSE)	150	ctDNA	Observational	Lung Cancer, esophageal cancer	NCT04014465
37.	Genotyping of Ebus-tbna supernant cell-free DNA in NSCLC (CELTICS)	66	ctDNA	Phase 2 interventional	Non-small cell lung cancer	NCT03865511
38.	Genomic profiling for metastatic solid tumor patients	50	cfDNA, supernatant free DNA (sfDNA)	Observational	Lung cancer	NCT04624373
39.	Osimertinib treatment on EGFR T790M plasma positive NSCLC patients (APPLE)	100	ctDNA	Observational	Solid tumor	NCT02215928
40.	Cell-free DNA methylation for epithelial ovarian cancer	156	cfDNA T790M mutation	Interventional	Non-small cell lung carcinoma	NCT02856893
41.	Study of circulating tumor DNA (ctDNA) kinetics in immuno-oncology (IO-KIN)	400	cfDNA	Phase 2 interventional	Ovarian cancer	NCT04651946
42.	Concordance between liquid and tissue biopsy	20	ctDNA	Observational	Head and neck squamous cell carcinoma	NCT04606940
43.	Concordance between liquid and tissue biopsy	120	CTCs ctDNA	Observational	Breast neoplasms	NCT04241237
44.	Tumor molecular profiling in early phase clinical trials	40	ctDNA	Observational	Advanced solid tumor, lymphoma	NCT04510766
45.	Multicenter Clinical Research utilizing Blood Plasma Derived Exosome for diagnosing Lung Cancer	470	Exosomes	Observational	Lung cancer	NCT04529915
46.	Monitoring breast cancer patients in Costa Rica using genetic markers of circulating tumor DNA	25	cfDNA	Observational	Metastatic breast cancer	NCT04163159
47.	Monitoring of blood sample of patients with EGFR-mutated lung cancer	250	ctDNA	Observational	Lung cancer	NCT02284633
48.	Dynamic monitoring of circulating tumor DNA in surgical patients with lung cancer (LUNGCA)	150	ctDNA	Observational	Lung cancer	NCT03317080
49.	A prospective study using circulatingcell-free DNA (cfDNA) in detecting RAS mutations in patients with advanced colorectal cancer	60	cfDNA	Interventional	Colon cancer	NCT04775862
51.	Prognosis and targeted therapy-associated molecular screening scheme for breast cancer patients in China	300	ctDNA, cfRNA	Observational	Breast cancer	NCT03792529
52.	Detecting circulating tumor cells (CTCs) and cell-free DNA (cfDNA) in peripheral blood for early detection, diagnostics and clinical application in breast cancer patients	210	cfDNA CTCs	Observational	Breast cancer	NCT03511859
53.	Evaluating the presence of urinary exosomes from clear cell renal cell carcinoma (PEP-C)	100	CD9+ and CA9+ exosomes	Observational	Clear cell renal cell carcinoma	NCT04053855
54.	ctDNA as a biomarker for treatment response in HNSCC (PECAN)	70	ctDNA	Interventional	Head and neck squamous cell carcinoma	NCT03540563
55.	Study of exosomes in monitoring sarcoma patients (EXOSARC)	30	Circulating exosomes	Observational	Sarcoma	NCT03800121
56.	Constituents of circulating extracellular vesicles: Biomarkers in colorectal cancer patients (ExoColon)	172	Circulating exosomes	Observational	Colorectal cancer	NCT04523389
57.	Liquid Biopsy to Distinguish Malignant From Benign Pulmonary Nodules and to Monitor Response to Therapy	171	DNA methylation	Observational	Non Small-cell Lung Cancer	NCT05462795
58.	The Feasibility of Dynamic Liquid Biopsy Monitoring During Neoadjuvant Treatment for EGFR-mutated NSCLC	300	ctDNA	Interventional	EGF-R Positive Non-Small Cell Lung Cancer	NCT06287593
59.	ctDNA in Cutaneous Squamous Cell Carcinoma	60	ctDNA	Observational	Cutaneous Squamous Cell Carcinoma	NCT06875609
60.	ctDNA Monitoring to Predict the Efficacy of Total Neoadjuvant Therapy for Rectal Cancer	100	ctDNA	Observational	Rectal Cancer	NCT06589388
61.	The circTeloDIAG: Liquid Biopsy for Glioma Tumor (circTeloDIAG)	150	ctDNA	Observational	Glioma	NCT04931732
62.	DANISH.MRD: Danish Assessment of Minimal Residual Disease by Liquid Biopsies	1600	ctDNA	Observational	Colorectal cancer	NCT06076811
63.	Liquid Biopsy in Head and Neck Cancer	30	ctDNA	Observational	Head and neck squamous cell carcinoma	NCT03926468
64.	Liquid Biopsy in Monitoring the Neoadjuvant Chemotherapy and Operation in Gastric Cancer	40	ctDNA, CTCs, cfDNA	Observational	Gastric Cancer	NCT03957564
65.	Study of Circulating Tumor DNA (ctDNA) Kinetics in Immuno-oncology (IO-KIN)	18	ctDNA	Observational	Head and neck squamous cell carcinoma	NCT04606940
66.	Longitudinal Tumor Burden Quantification Using Circulating Tumor DNA in Metastatic Lobular Breast Cancer (LBC-Monitor)	20	ctDNA	Observational	Breast cancers	NCT06666439
67.	Implementation of Up-front ctDNA Into Lung Cancer Care and Development of Liquid Biopsy-based Decision Support Models—LM2 Study (LM2)	800	ctDNA, CTCs	Observational	Lung cancer	NCT06105177
68.	Molecular Profiling and Dynamic Changes of ctDNA in Unresectable Locally Advanced NSCLC	80	ctDNA	Observational	Lung cancer	NCT05641870
69.	SIBYL: obServation of Therapy Response With lIquid BiopsY evaLuation	440	ctDNA	Observational	Breast Cancer Colorectal Cancer Non-small Cell Lung Cancer	NCT05935384
70.	LIBERTY: Liquid Biopsy to Diagnose and Monitor CNS Involvement in High-risk B Cell Non-Hodgkin Lymphoma (SAKK 38/23)	64	ctDNA	Interventional	Non-Hodgkin B-cell lymphoma	NCT06090162
71.	Liquid Biopsy to Enable Diagnostics and Monitoring for Immune-mediated Lymphoproliferative Disorders (LIMPID)	20	ctDNA	Observational	Lymphoproliferative Disorders	NCT05803616
72.	Liquid Biopsies in Pediatric Solid Tumors	320	cfDNA	Observational	Non-CNS Malignant Pediatric Solid Tumors	NCT05068583
73.	Temozolomide and Irinotecan in Patients With MGMT-Silenced Colorectal Cancer After Adjuvant Chemotherapy (ERASE-TMZ)	35	ctDNA	Interventional	Colorectal Cancer	NCT05031975
74.	MEchanisms of Resistance in EGFR Mutated Non-pretreated Advanced Lung Cancer Receiving OSimErtib (MELROSE)	150	ctDNA	Interventional	Non-small Cell Lung Cancer	NCT03865511
75.	Monitoring luminAl Breast Cancer Through the Evaluation of Mutational and epiGeNEtic alteraTIons of Circulating ESR1 DNA (MAGNETIC1)	164	Circulating ESR1 DNA	Interventional	Hormone Receptor-Positive Breast Carcinoma, Breast Neoplasms, Neoplasms, Breast Breast Diseases, Antineoplastic Agents, Aromatase Inhibitors, ESR1 Gene Mutation	NCT05814224

.

3. Nanomaterials Utilized for Liquid Biopsy Applications

Nanotechnology-based techniques have developed rapidly in recent years and, compared to traditional analytical methods, offer advantages such as high sensitivity, simplicity, multiplex detection, automation, and miniaturization, supporting their potential use in clinical practice. Figure 3 compared the workflows of conventional and nano-based liquid biopsy. Nanoparticles such as gold, carbon-based nanomaterials like graphene oxide and quantum dots (QDs), as well as silver, silica, magnetic, and iron oxide nanocrystals hold tremendous potential due to their unique optical, electronic, structural, and magnetic properties, which aid in the sensitive detection of liquid biopsy biomarkers [67]. Some of the nanotechnological applications for detection of liquid biopsy are tabulated in

Table 3. Nanotechnology in detection of liquid biopsy biomarkers.

S. No	Nanotools	Detection Method	Markers	Applications	Advantages	Ref.
CTC Detection
1.	Gold nanorods and magnetic nanoparticles	Photoacoustic detection	Folic acid (FA)	-	High sensitivity	[45,68]
2.	Multifunctional magnetic upconversion nanoparticle (MUNP)	Luminescence imaging	EpCAM	Lung cancer patients	Highly sensitive for detecting tumor cells	[69]
3.	Carbon nanotubes	Impedimetric detection	EpCAM	Liver	Good conductivity	[70]
4.	Si nanopillars (SiNP)	Optical detection	EpCAM	-	Specificity	[46,71]
5.	Microchip- based immunomagnetic	Immunomagnetic microfluidic device	EpCAM	Breast, prostate and lung cancer	Sensitivity and specificity	[72,73]
6.	CTC-iChip	Immunomagnetic microfluidic device	EpCAM	Prostate, breast, pancreas, colorectal and lung cancer	Highly sensitive and portable	[74]
7.	Magnetic ranking cytometry (MagRC)	Immunomagnetic microfluidic device	EpCAM	Prostate	Accuracy and cost-effective	[75]
EV Detection
8.	ssDNA attached g-C3N4 nanosheets	Colorimetric detection	CD63	-	Highly sensitive detection	[76]
9.	Nanotetrahedron-assisted aptasensor	Electrochemical assay	-	-	Highly sensitive and portable	[43]
10.	AuNPs	Lateral flow immunoassays	CD9, CD81 and CD63	Metastatic melanoma	Inexpensive, simple, fast	[77]
11.	Fe3O4 NPs	Colorimetric	EpCAM	-	Accurate, highly sensitive, and specific	[78]
12.	Graphitic carbon nitride nanosheets	Colorimetric	CD63	Breast cancer	Fast, highly sensitive and cost-effective	[79]
13.	AuNPs	Colorimetric	LMP1	Nasopharyngeal carcinoma	Inexpensive, simple, fast, and easy handling	[80]
14.	AuNR	Colorimetric	CD63 and phospholipid membrane	Breast cancer	High sensitivity	[81]
15.	CuO NPs	Fluorescence	CD63	Liver cancer	Convenient, efficient, and cost-effective	[82]
16.	AuNP-linker/complementor	Fluorescence	CD63	Liver cancer	Inexpensive, simple, fast, and easy handling	[83]
17.	QDs	Fluorescence	CD63	Breast cancer	Convenient, Efficient, and cost-effective	[84]
18.	MoS2-MWCNT	FRET (quenching fluorescence)	CD63	Breast cancer	High sensitivity	[85]
19.	GO	FRET (quenching fluorescence)	CD63, EpCAM, PDGF	Breast cancer, gastric cancer, cervical cancer and liver cancer	Convenient, efficient, and cost-effective	[86]
20.	MXene nanosheets	FRET (quenching fluorescence)	CD63	Breast cancer, ovarian carcinoma and liver cancer	Accurate, highly sensitive, and specific	[87]
21.	Au-NPFe2O3NC	Electrochemical (voltametric)	CD63	Placental choriocarcinoma	Convenient, efficient, and cost-effective	[88]
22.	CdSeQD	Electrochemical	HER-2 and FAM134B	Colorectal carcinoma	Inexpensive, simple, fast, and easy handling	[89]
23.	AgNPs and CuNPs	Electrochemical	EpCAM and PSMA	Prostate cancer	Fast, highly sensitive and cost-effective	[90]
24.	Ti3C2MXenes	Electrochemiluminescence	EpCAM	Melanoma, breast cancer, and liver cancer	High sensitivity	[91]
25.	Au@Ag NRs	SERS	CD63, HER2	Breast cancer, lung fibroblasts	Fast, highly sensitive and cost-effective	[92]
26.	Nanoholes patterned in a Au film + Au nanostars	Surface plasmon resonance spectroscopy (SPR)	CD63	Ovarian cancer	Accurate, highly sensitive, and specific	[29]
27.	Magnetic–electrochemical exosome device (iMEX)	Electrochemical assay	CD63, EpCAM, CD24, and CA125	-	Sensitive, portable, high-throughput	[93]
28.	Nanomaterials using Raman Scattering	Surface-enhanced Raman scattering (SERS)	Folic acid	-	Prominent sensitivity and specificity	[94]
29.	AuNRs-UCNPS (paper)	Luminescence resonance energy transfer (LRET)	CD63	Liver cancer	-	[95]
30.	Nanoplasmonic sensor	Periodic nanohole array	CD24 and EpCAM	97% for the ascites sample of patients having ovarian cancer	High sensitivity	[96]
		Periodic nanohole array	EGFR, EPCAM, MUC1, GPC1 and WNT2	86% for pancreatic ductal adenocarcinoma	Accurate, highly sensitive, and specific	[97]
		Nanoplasmon-enhanced scattering assay	CD81, CD63, CD9, and EphA2	Plasma of pancreatic cancer patients	Fast, highly sensitive and cost-effective	[98]

.

3.1. Gold Nanostructures

Gold nanoparticles are commonly utilized for various applications of liquid biopsy. Their distinctive electrical and optical features, biocompatibility, ability for conjugation with various biomolecules, high stability, and large surface-area-to-volume ratio make them a unique platform for the development of versatile biosensors [99]. Additionally, they possess several analytical applications in sensing which include SPR, fluorescence resonance energy transfer (FRET), surface-enhanced Raman spectroscopy (SERS), lateral flow assays, and colorimetric and fluorescence methods [99,100].

3.1.1. MicroRNAs

Gold nanoparticles are utilized for detecting microRNA (miRNA), which are associated with the emergence of cancer. In one study, gold nanoparticles (AuNPs) were conjugated with DNA probes and allowed to hybridize with the miRNA target in the presence of Mg²⁺, resulting in the formation of a DNAzyme that digests the hybrids, generating fluorescent fragments. This process presented a limit of detection (LOD) of 50 fM [100]. Nossier et al. revealed a colorimetric method for detecting miRNA. This method was dependent on the stability of AuNPs stimulated by the hybridization of the miRNA target with short complementary DNA probes [101,102]. Moreover, lateral flow strips were employed to detect miRNA using AuNPs as reporters for visual detection, based on a sandwich-type hybridization assay on a nitrocellulose membrane [103]. Additionally, AuNPs coupled with rolling circle mechanism of replication for dual signal enhancement were utilized for simultaneous detection of two miRNAs [104]. AuNPs can also be conjugated with detection probes and many horseradish peroxide (HRP) molecules, which aid in enhancing the optical output by adding a chromogenic substrate [105]. Mohammad Niaei et al. developed a three-way junction RNA structure for detection of miRNA which contained a methylene blue-altered hairpin structure at one end (which works as a sensing moiety); the other ends were hybridized with DNA-barcoded AuNPs for amplifying the signals. Upon hybridization with target miRNA, the hairpin moiety was opened with successive hybridization onto a DNA-modified platinum/gold electrode, resulting in oxidation of methylene blue. This method required a very small amount of sample i.e., 4 μL, and showed an LOD of 135 Am, which is 230 times greater than RT-PCR [106]. Fredj et al. developed a voltammetric detection method for miRNA by hybridizing it with biotinylated beacons linked to gold nanoparticles, which were then captured via avidin–biotin interaction. This method can detect miRNA at concentrations as low as 4 fM [107].

AuNPs in association with magnetic beads and strand displacement amplification (SDA) were used for detecting miRNA. In this method, AuNPs coupled with magnetic beads were allowed to conjugate with the DNA probe complementary to the target miRNA sequence via a sandwich-like hybridization. The target miRNA was hybridized with its complementary probe via strand displacement and resulted in the release of AuNPs from the magnetic beads. The proportion of free AuNPs is related to the concentration of target miRNA and was observed through dark-field microscopy [108]. Moreover, SPR has been utilized to detect miRNA by hybridizing it with complementary DNA probes immobilized on the sensor chip. The resulting hybrids were captured with a biotinylated antibody that recognizes DNA-RNA complexes, and neutravidin-conjugated AuNPs were used for signal enhancement [109].

3.1.2. Circulating Tumor DNA

Gold nanorods and AuNPs conjugated with peptide nucleic acid (PNA) probes on a SPR sensor have been utilized to detect at least two nanograms of synthetic single-stranded ctDNA/mL within ten to fifteen minutes [110]. A colorimetric assay based on a three-way target CHA was employed for the detection of ctDNA. Following hairpin assembly, DNA fragments induced the aggregation of gold nanoparticles, causing a color change from red to blue [67]. One more colorimetric detection method was employed, where accumulation of unlabelled AuNPs took place and isolated ctDNA from blood samples bound to AuNPs resulted in a visible change in color [111]. Lin et al. utilized an SERS nanosensor for signal amplification in detecting ctDNA based on silica-coated Au nanorods. This method utilized metal-carbonyl SERS labels for enhancing the sensitivity of detection [67]. Additionally, to detect mutations in ctDNA, a lateral flow strip test was designed using AuNPs. In this method, the targeted DNA was allowed to amplify via allele-specific PCR with a specific oligonucleotide tail at one end. The resulting amplified DNA was placed on the strip and examined by AuNPs through hybridization with the oligonucleotide tail [112].

3.1.3. Circulating Tumor Cells

To detect CTCs, a dual amplification methodology was utilized by employing AuNPs and rolling circle amplification (RCA). Long ssDNA sequences were generated by RCA which were then captured by DNA-AuNPs conjugates, and the arrested AuNPs were analyzed using inductively coupled plasma mass spectrometry (ICP-MS) [113]. Moreover, the targeting of CTCs in blood samples was performed by employing aptamer-modified gold nanofilms (10–100 nm) utilizing pulsed-laser desorption/ionization mass spectrometry (LDI-MS). This method was able to identify at least ten cancer cells in a blood sample [114]. For visual detection of CTCs, an aptamer-AuNPs-based strip was utilized in which the positive signal at the strip was induced by other biotinylated aptamer conjugated with immobilized streptavidin at the test zone by interacting with CTC–aptamer–gold nanoparticle complexes. This method was able to detect at least 4 × 10³ CTCs with the naked eye and as few as 800 CTCs using a portable reader, all within a few minutes [115].

3.2. Graphene Oxide (GO) Nanostructures

Two-dimensional graphene oxide (GO) is a great carbon-based nanomaterial with several advantages in bioimaging, biosensing, and drug delivery. Various spectrometric and electrochemical biosensors have been constructed using GO for biosensing. GO offers a good surface area which helps in providing a new platform for signal enhancement, possesses good electrical properties, and offers distinct functional groups for bioconjugation. GO provides excellent quenching ability and adsorption ability for ssDNA which can be utilized for various fluorescent applications [113].

3.2.1. MicroRNAs

GO can be employed for detecting miRNA by developing a GO-fluorescence-based assay which involves rolling circle amplification and ssDNA [116]. Treerattrakoon et al. proposed a multiplex miRNA-sensing method by utilizing various ssDNA, RCA, and GO, which was used as a fluorescence quencher [117]. Further, the GO surface was covalently coupled with the ssDNA, and fluorescently labelled DNA probes were used, containing two segments among which one is complementary to the immobilized DNA sequence and the other is complementary to the miRNA sequence. When DNA probes are in close proximity to GO, due to hybridization with single-stranded DNA sequences on its surface, fluorescence quenching occurs. However, upon addition of the miRNA target, the fluorescence is restored, with the recovered signal correlating to the amount of miRNA present [118]. Avila et al. investigated a new method for detecting miRNA by employing labeled GO/ssDNA complexes that are coated with gold nanowires that have the potential to capture cancer cells [119]. Ryoo et al. utilized the same mechanism by using a labelled peptide nucleic acid (PNA) probe and GO for detecting miRNA, and the fluorescence signal was recovered when the labelled PNA probe or ssDNA bound to the targeted miRNA [119,120]. Duplex molecular beacons are utilized for detecting miRNA colorimetrically where the target miRNAs are hybridized using strand displacement, causing the release of peroxidase-mimicking DNAzyme, which was collected on the surface of GO. The released DNAzyme is responsible for catalyzing a colorimetric reaction and provided an LOD of 12.9 nM [121]. The electrochemical sensing of miRNA included immobilization of DNA probe on an electrode. The target miRNA was allowed to hybridize with specified probes, and GO was accumulated on the hybrids, aggregating methylene blue dye which can reduce the pulse voltametric signal [122]. In this method, duplex-specific nucleases were used for the recycling of target and amplifying the signal. Similarly, an electrochemical sensing method for detecting miRNA was developed using the same approach by utilizing Prussian Blue nanoparticles on the surface of GO [123].

3.2.2. Circulating Tumor Cells

CTCs can be detected by employing an antibody-modified reduced GO, and the captured cells can be observed by immunostaining, thus detecting 2 CTCs/4 mL blood [124]. An aptasensor was developed by utilizing tetra-(4-aminophenyl) porphyrin-dependent reduced GO for the electrochemical identification of a minimum of 10 CTCs/mL [125]. Xiao et al. developed an aptasensor using hairpin aptamer probes, nicking endonuclease, linker DNA probes with dye labelling, and GO to detect as least 25 CTCs in a blood sample [126]. Furthermore, GO modified with AuNPs can be used for electrochemical detection of as few as 40 CTCs/mL by employing β-cyclodextrin–AuNPs/GO complexes or ferrocene–aptamer/Ru(bpy)₃²⁺ systems. These electrochemical aptasensors can be reused for six to seven cycles [127].

3.3. Quantum Dot (QD) Nanostructures

QDs offer numerous advantages for use in liquid biopsy. They possess excellent optical properties, such as broad excitation spectra, narrow emission peaks, high photostability, and strong fluorescence, all of which contribute to enhanced biosensing sensitivity compared to conventional fluorescent dyes. The use of QDs for the detection of miRNA generally employs lateral flow assays, electrochemiluminescence, and FRET. An electro-chemiluminescent biosensor was proposed for detecting miRNA using target recycling and a Zn²⁺-driven DNA rolling mechanism for amplifying the signals. The DNA nanomachine was developed by AuNPs conjugated with DNA probes which were hybridized and moved along the bound complementary DNA probes with Zn²⁺ recognition sites over the electrode surface. On the electrode surface, CdS@Mn QDs were used for electro-chemiluminescent signalling with signal quenching by ferrocene. The electro-chemiluminescent signal was restored for detecting miRNA [128]. Moreover, for the photoelectrochemical detection of miRNA, a DNA tetrahedron was developed as a nanocarrier for immobilizing CdTe QDs. This method utilized enzyme-assisted target cycling amplification and a QD–dye complex for detecting miRNA with an LOD of as low as 17 Am [129,130]. A dual-channel ratiometric nanoprobe was constructed for imaging and detecting miRNA by using molecular beacons with fluorescent dye and MoS2-QDs. FRET efficiency was low when the molecular beacon was coupled with QDs, but once miRNA hybridized with molecular beacons, the distance between dye and QDs increased, providing a strong FRET signal [131]. QDs are also used for detecting CTCs. Xie et al. developed a multifunctional nanobioprobe by coating the surface of QDs with alginate activated by Ca²⁺, followed by immobilization of biotinylated anti-EpCAM antibodies. This multifunctional nanoprobe has the potential for specific cell capture [132]. Similarly, Min et al. developed anti-EpCAM-modified QDs to capture CTCs and secondary anti-IgG-magnetic beads for linking the attached cells. They showed that the fluorescence intensity of QDs was equivalent to the captured number of cells. This method possesses high target efficiency and an easy quantification method for CTCs [133].

3.4. Copper Nanoparticles (CuNPs)

Metallic nanoclusters possess distinctive features which can be utilized in various applications of liquid biopsy. They are smaller in size and exhibit excellent conductivity, quantum effects, intrinsic fluorescence, and ferromagnetic and chirality features. Various copper nanostructures have peroxidase-like activity, and in the presence of hydrogen peroxide, they can oxidize the chromogenic substrate. Copper was used in the form of DNA-Cu nanoclusters by utilizing peroxidase-like catalytic activity to visually detect concentrations of miRNA [134]. Xu et al. designed fluorescence-based detection of miRNA by using duplex-specific nucleases (DSNs) that stimulate amplification of the target, thus leading to terminal deoxynucleotidyl transferase-mediated synthesis of polyT-CuNPs, which can carry out fluorescence-based detection of miRNA [135]. Additionally, the synthesis of copper nanoblocks linked with DNA probes was followed by hybridization, and finally, magnetic isolation resulted in fluorescent and electrochemical detection of miRNA in solution [136]. It has been shown that the target miRNA hybridized with a hairpin bound to an electrode started a hybridization chain reaction (HCR). The copper nanoclusters were generated on AT-rich dsDNA produced as a result of HCR and provided an electro-chemiluminescent signal for detecting miRNA [137].

3.5. Silver Nanoparticles (AgNPs)

Silver nanoparticles have been extensively employed for enhancing signals in SPR, SERS, and electrochemical detection because of their easy oxidation process and greater extinction coefficient value. Silver nanoclusters were employed for detection of miRNA by triggering catalytic hairpin assembly utilizing two hairpins. One of the hairpins consisted of silver nanoclusters, and the second hairpin bore G-rich DNA sequences. CHA took place when, upon hybridization of target miRNA with first hairpin—which was followed by close association among silver nanoclusters and G-rich regions, which increased the fluorescence of nanoclusters— as little as 0.3 nMmiRNA was detected [138]. Moreover, an easy method was developed for detecting miRNA in which silver nanoclusters were coupled with a DNA probe, which was allowed to hybridize with target miRNA. The negatively charged DNA probe interacted with positively charged gold nanoparticles, thereby quenching the fluorescence of silver nanoclusters. This was followed by hydrolysis of the RNA-DNA hybrid by duplex-specific nucleases, which removed the gold nanoparticles, leading to fluorescence recovery and enabling miRNA detection [139]. Similarly, silver nanorods were used for ctDNA detection via an SERS system combined with target-triggered enzyme-free recycling and a signal enhancement step involving various reporters, enabling detection of ctDNA at 40.4 aM [140]. For the detection of CTCs, silver nanoprisms in association with magnetic iron oxide nanoparticles were used by employing magnetic enrichment and SERS detection system [141].

3.6. Silica Nanoparticles (SiNPs)

Silica nanoparticles (SiNPs) are widely used in various analytical detection techniques due to their ability to encapsulate multiple molecules within a single nanoparticle. They can also encapsulate dye or reporter molecules, enhancing the detection process. SiNPs have been employed for miRNA detection by coupling them with fluorescent dye-labeled DNA probes. The fluorescence of the dye is initially quenched by the SiNPs and is restored upon hybridization with the target miRNA [142]. Similarly, a DNA–SiNPs complex was used for miRNA detection. A fluorophore is attached to the immobilized DNA probe, while a quencher molecule is attached to a complementary probe. When the quencher-carrying probe hybridizes with the immobilized DNA probe, fluorescence is quenched. This fluorescence is restored when miRNA displaces the quencher probe upon hybridization [143].

3.7. Iron and Magnetic Nanostructures

Iron and magnetic nanoparticles have been extensively used in biosensing and detection due to their ability to enhance signals, thereby improving the detectability of biomarkers. Additionally, their peroxidase-like activity enables colorimetric detection. Au-coated paramagnetic nanoparticles have been utilized for electrocatalytic determination of miRNA through SERS, employing conjugated DNA probes [144,145]. Additionally, magnetic Fe@SiO₂ nanoparticles were attached to DNA probes, while gold nanoparticles were conjugated with complementary DNA probes for detecting ctDNA. The silica coating prevented oxidation of the magnetic nanoparticles and enhanced their biocompatibility and solubility. When both nanoparticles come into close proximity through hybridization, magnetic isolation is performed, followed by measuring gold concentration via inductively coupled plasma mass spectrometry (ICP-MS), enabling detection of ctDNA at levels as low as 0.1 pg/mL [146]. Magnetic Fe₃O₄ nanoparticles conjugated with silicon nanoparticles linked to anti-MUC1 aptamers have been utilized to detect cancer cells at concentrations as low as 100 cells/mL. Similarly, Fe₃O₄ nanostructures coupled with cross-linked PEG have been employed for detecting CTCs [147].

4. Limitation

While nanotechnology holds great promise for advancing liquid biopsy diagnostics, several limitations must be carefully addressed to ensure successful clinical translation. One major concern is cytotoxicity, particularly with inorganic nanomaterials such as quantum dots and silver nanoparticles, which can induce oxidative stress or release toxic ions. Additionally, many nanoparticles exhibit poor biodegradability and may accumulate in organs such as the liver and spleen, raising safety concerns for long-term use. In diagnostic assays, off-target effects and non-specific binding can reduce sensitivity and specificity, potentially leading to false-positive or false-negative results, especially in complex biological fluids. Moreover, challenges in scalability and reproducibility continue to hinder regulatory approval, as clinical-grade nanoparticles must meet stringent quality standards under Good Manufacturing Practices (GMPs). To date, only a limited number of nano-enabled liquid biopsy platforms have received FDA or EMA approval, largely due to unclear regulatory pathways and the need for extensive clinical validation. Additionally, the high cost and complexity of some nanomaterial-based systems may limit their widespread adoption, particularly in low-resource settings. Addressing these challenges through the development of biocompatible materials, standardized manufacturing protocols, and collaborative translational research will be critical to realizing the full potential of nanotechnology in cancer diagnostics.

5. Conclusions

The emerging field of liquid biopsy has opened up new avenues for cancer diagnosis, with significant clinical applications in personalized oncology. Advances in nanotechnology have greatly enhanced the detection, characterization, and sensing of liquid biopsy biomarkers such as circulating tumor cells (CTCs), cell-free nucleic acids (cfNAs), circulating tumor proteins (CTPs), microRNAs (miRNAs), and extracellular vesicles (EVs). Nanomaterials provide excellent platforms for advanced cancer diagnostics, treatment evaluation, and monitoring of disease progression, offering high sensitivity, selectivity, and rapid analysis. Nanotechnology has evolved substantially from the use of individual nanostructures or nanomaterials to the development of multifunctional, integrated nano-based devices and platforms. Future directions in this field include the development of point-of-care nano-enabled liquid biopsy devices, integration with artificial intelligence for data interpretation, and the design of personalized nanosensors for real-time monitoring. Emerging research is also exploring biodegradable and stimulus-responsive nanomaterials that can adapt to dynamic tumor environments, thereby improving diagnostic precision and safety. However, the routine application of nanotechnology-based liquid biopsy in clinical settings remains a work in progress. Ethical considerations—such as data privacy, equitable access to advanced diagnostics, and the long-term biocompatibility of nanomaterials—must be addressed to ensure responsible clinical translation. In parallel, regulatory frameworks must evolve to guide the safe and effective implementation of these technologies. Finally, more extensive research and collaborative efforts are urgently needed to promote translational research and facilitate the clinical adoption of nanotechnology-enabled liquid biopsy tools.