Magnetic-assisted manipulation of rare blood cells for diagnosis: A systematic review

Abstract

The precise isolation and analysis of rare cells from blood are crucial for biomedical research and clinical diagnostics. This review examines recent advancements in magnetic-based separation techniques, focusing on their efficiency in capturing rare cells such as circulating tumor cells (CTCs), circulating fetal cells, and diseased red blood cells (RBCs). These methods use magnetophoresis under external magnetic fields for highly specific isolation with minimal contamination, offering advantages over traditional techniques in speed, cost-effectiveness, and robustness. Magnetic separation is categorized into label-based methods, which use immunomagnetic nanoparticles (IMNs) to target specific cell markers, and label-free methods, which exploit differences in magnetic susceptibility. Both approaches have achieved up to 99% efficiency in isolating diseased RBCs and CTCs. However, challenges remain in improving purity, scalability, and clinical applicability. A key limitation of label-based methods is the need to detach cells from magnetic beads without compromising viability. Label-free technologies, such as magnetic levitation, enable ligand-free separation based on density and susceptibility. Future research should focus on optimizing paramagnetic media, integrating machine learning for enhanced accuracy, and developing high-gradient magnetic fields (~1,000 T/m) to improve efficiency. Advancements in IMNs with stronger magnetic properties will further enhance separation performance, driving clinical translation.

Graphical Abstract

This systematic review evaluates recent advancements in magnetic-based separation techniques for isolating rare cells, including circulating tumor cells and diseased red blood cells. Label-based and label-free methods achieve up to 100% and 97% capture efficiency, respectively, highlighting their potential for improving disease diagnostics and personalized treatments.

1. Introduction

A crucial procedure for the in-depth study of diseases in biomedical and clinical research as well as for disease diagnosis is the separation and analysis of certain types of cells from complex biological fluids with minimal contamination from other non-target cells.(Frenea-Robin & Marchalot, 2022b; Leong et al., 2018) Blood and its constituents are one of the most common biological samples for the analysis of cells in the field of diagnosis. Nucleated cells at a concentration lower than 1,000 cells in one millimeter of blood sample are known as “rare cells”.(Chen et al., 2014; Schreier & Triampo, 2020; Smejkal et al., 2023) These cells are vital for the diagnosis and prognosis of many diseases as they reveal important information about an individual’s health status.(Chen et al., 2014) Some significant groups of rare cells include circulating tumor cells (CTCs) for cancer prognosis and circulating fetal cells for prenatal testing. In one milliliter of blood, the number of these cells is estimated to be 1–1,000 for CTCs and 1–2 for circulating fetal cells.(Chen et al., 2014; Kavanagh et al., 2010) Within the realm of cancer, the separation and analysis of CTCs is of paramount importance for studying the disease progression. CTCs, first discovered by an Australian pathologist, Thomas Ashworth, in 1869, have similar morphological features and genetic expressions to the primary tumor and have been used as a diagnostic and prognostic marker for cancer.(Ashworth, 1869; Ju et al., 2022; Smejkal et al., 2023) On the contrary, circulating fetal cells have shown to be very useful for non-invasive fetal testing. In 1959, these cells were first reported in maternal blood.(Singh et al., 2017; Tang et al., 2022) They are shed from trophoblast or umbilical cord blood and play a significant role in prenatal diagnosis for the screening of fetal diseases and the acquisition of a fetal genetic profile.(Holzgreve et al., 1992; Huang et al., 2020; Sabbatinelli et al., 2021; Yamanishi et al., 2002). Additionally, certain conditions can also be diagnosed or managed by inspecting a small amount of red blood cells (RBCs) in a patient’s sample. For all these applications, isolation of the cells of interest from the vast number of cells in a blood sample at a high purity and efficiency is needed. However, while much of the existing literature has focused on CTC isolation, recent advancements have broadened the scope of isolation systems to include RBCs, which have proven diagnostic potential, especially in conditions like sickle cell disease, anemia, and malaria.(Arishi et al., 2021; Nam et al., 2013)

Over the course of 30 years, magnetic-based manipulation and separation techniques have garnered significant attention in the capture and analysis of cells for the diagnosis of multiple conditions. Under the application of an external magnetic field generated by permanent magnets or electromagnetic coils, the target cells can be isolated from the blood in a phenomenon known as magnetophoresis.(Gómez-Pastora, Moore, et al., 2022; Xian Wu et al., 2022; Zborowski & Chalmers, 2011; Zborowski et al., 2002) The advantages of magnetic-based isolation techniques include high throughput, economical cost, less energy-intensive, and no detrimental effects on the cells since they are essentially non-invasive.(Leong et al., 2016; Leong et al., 2018) Magnetic-based separation techniques can be divided into two categories: label-based techniques and label-free techniques.(Iranmanesh & Hulliger, 2017)

As shown in Figure 1, label-based separation techniques involve the capture of cells with immunomagnetic micrometer- (0.1–50 μm) and nanometer-(10–100 nm) sized magnetic particles (IMNs) that target specific surface receptors of the cells.(Gómez-Pastora et al., 2020; Plouffe et al., 2015) These particles are composed of an iron oxide core, such as magnetite (Fe₃O₄) or maghemite (γ-Fe₂O₃), with a biocompatible (usually polymer) coating.(Wu et al., 2024; Wu et al., 2023) The IMNs can then be functionalized with antibodies that target specific cells, and the magnetically labeled cells can then be selectively isolated under a magnetic field gradient, also known as immunomagnetic-based separation. Immunomagnetic-based separation techniques have gained popularity in recent decades, as these techniques are simple, are not time-consuming, and do not involve any centrifugation or filtration procedures that can lead to the loss of viable cells.(Gómez-Pastora, Moore, et al., 2022)

Figure 1.

Schematic illustrating the principles of labeled and label-free magnetophoresis.

For CTC isolation using IMNs, the most targeted surface antigen is the epithelial cell adhesion molecule (EpCAM) because this antigen is expressed on the surfaces of most epithelial CTCs.(Bankó et al., 2019) However, after CTCs undergo epithelial-to-mesenchymal transition, the EpCAM expression decreases, so CTCs with low or no EpCAM expression cannot be detected, reducing the capture rate significantly.(Hu et al., 2021; van der Toom et al., 2016) Therefore, recent efforts in the field have been focused on developing novel IMNs targeting CTCs with non-EpCAM functional groups to selectively capture those cells with low/no EpCAM expression. Among the other biological molecules that can be conjugated to IMNs for the detection of CTCs there are other antibodies, peptides, or aptamers that can efficiently target cancer-specific receptors (e.g., Herceptin for breast cancer), mesenchymal markers (N-cadherin for breast cancer), or receptors overexpressed on cancer cells (folic acid).(Haghighi et al., 2019; Li et al., 2018; Pipatwatcharadate et al., 2023; Wang et al., 2019) After isolation, CTCs can be further identified and analyzed molecularly, as demonstrated in Figure 2, to provide insights into their biological characteristics and potential therapeutic targets. With regards to circulating fetal cells, there is no antibody that can target a specific surface antigen. However, several studies have been carried out where circulating fetal cells have been successfully captured by anti-CD71 antibody-functionalized IMNs.(Collarini et al., 2001; Prieto et al., 2001)

Figure 2.

Schematic demonstrating the immunomagnetic capture of CTCs followed by identification and molecular analysis of CTCs.

However, labeled techniques have limitations, such as limited specificity depending on labeling antibodies, low cell recovery, low efficiency in separating complex mixtures, and potential cell damage due to the separation of cells from IMNs for further downstream analysis.(Frenea-Robin & Marchalot, 2022b; Plouffe et al., 2015) In recent years, there has been a growing interest in developing label-free separation techniques. This technique offers unique advantages such as simplicity, faster processing time, high throughput, and minimal cell perturbation.(Choe et al., 2024; Gossett et al., 2010)

Label-free separation techniques exploit the differences in the physical properties of the cells, such as magnetic susceptibility, which act as a marker for cell isolation.(Wyatt Shields Iv et al., 2015; Zborowski & Chalmers, 2011) A cell’s magnetic susceptibility is its ability to be magnetized under the application of an external magnetic field.(Frenea-Robin & Marchalot, 2022b) Most cells and biological materials are diamagnetic. However, certain cells, such as deoxygenated RBCs and methemoglobin RBCs, exhibit paramagnetism due to a pair of unpaired electrons from the ionic bonds between the iron in the heme group and the histidine side chains of the polypeptide chains within the hemoglobin (Hb) molecule.(Barua et al., 2023; Gómez-Pastora et al., 2021; M. Weigand et al., 2022; Wyatt Shields Iv et al., 2015) The change in the paramagnetic properties of RBCs due to disease is recently gaining attention in the diagnosis of several conditions, such as sickle cell disease, anemia, and malaria.(Hackett et al., 2009; M. Weigand et al., 2022) For instance, Weigand et al. determined that sickle RBCs have higher magnetic susceptibilities than healthy RBCs, which can be attributed to their lower oxygen-binding ability compared to healthy cells.(M. Weigand et al., 2022) Analogously, malaria-infected RBCs have a greater magnetic susceptibility than healthy RBCs, as the parasites convert Hb to hemozoin, which contains iron (III) or Fe³⁺.(Leong et al., 2018) Due to the presence of Fe³⁺, infected RBCs have a stronger paramagnetic character than healthy cells. Magnetophoresis can then be utilized to fractionate infected cells from biological samples to diagnose and treat malaria.(Barua et al., 2023; Gómez-Pastora et al., 2018).

By exploring both techniques (i.e., labeled and label-less magnetophoresis), we provide a comprehensive review that addresses both CTC and RBC isolation, recognizing their diagnostic potential using magnetic separation techniques. By addressing the magnetic separation of both CTCs and RBCs, this paper integrates numerous accounts of magnetic separation techniques for rare blood cells and their diagnostic applications across a variety of diseases. This review is, to the best of our knowledge, the first to provide an integrated discussion of both cell types using both technologies, offering new perspectives on their respective roles in disease detection.

In this systematic review, we meticulously examine selected articles from current literature (2019–2023) that focus on the development and optimization of magnetophoresis, and manipulation techniques employed in the diagnosis of diseases, mainly focusing on the isolation of CTCs, circulating fetal cells, and infected and anemic RBCs. After this introduction, Section 2 highlights the methodology followed for the selection of studies. Sections 3 and 4 review the current strategies proposed for the magnetic-assisted diagnosis of conditions achieved by targeting the labeled or label-free manipulation of CTCs, fetal cells, and RBCs. This review concludes with current challenges in this field in Section 5 and with the main conclusions and further directions in the magnetic-assisted diagnosis in Section 6.

2. Methodology

2.1. Information Sources and Search Strategy

A comprehensive literature search was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines to identify relevant studies on the application of magnetophoresis employed in the diagnosis of diseases, particularly focusing on the isolation of CTCs, circulating fetal cells, and RBCs for several types of anemia and malaria infection detection. The search was performed using two electronic databases, PubMed and Web of Science, which were chosen for their extensive coverage of biomedical and scientific literature.

The search string was carefully designed to capture key studies in the field, including magnetic separation techniques, target cells (CTCs, fetal cells, and RBCs), and sample type (blood). The employed search string contained terms for magnetophoresis (magnet* OR magnetophoresis OR immunomagnetic), isolation and diagnosis (isolat* OR sort* OR separat* OR diagnos* OR identif* OR enrich*), and the target cells under consideration (“red blood cells” OR erythrocytes OR RBCs OR “iron deficiency” OR malaria OR anemia OR sickle OR “cancer cells” OR “tumor cells” OR CTCs OR “Circulating Tumor Cells” OR “fetal cells”). The search string included both free-text terms and controlled vocabulary (e.g., MeSH terms in PubMed) to maximize the retrieval of relevant articles. The asterisk (*) was used as a wildcard symbol to account for variations in word endings and spellings. The Boolean operators “AND” and “OR” were used to combine the search terms and define the relationships between the key concepts. To focus on the most recent and relevant studies, the search was limited to articles published within the last five years, between 2019 and 2023. Additionally, the search was restricted to articles written in English to ensure that the included studies could be easily understood and analyzed. These restrictions were applied using the built-in filters available in the electronic databases.

2.2. Study Selection and Data Extraction

The initial search yielded a total of 6,296 records, with 3,555 results from PubMed and 2,741 results from Web of Science. The results of the search were imported into MS Excel (Microsoft 365, version 2405 Build 16.0.17628.20006) software for duplicate removal and screening. The titles and abstracts screening were performed independently and resulted with 498 records from Web of Science and 355 records from PubMed. Studies were included if they focused on the application of magnetophoresis for the detection of disease biomarker cells, emphasizing the enrichment of CTCs, fetal cells or parasitized/anemic RBCs. Disagreements between reviewers were resolved through discussion or by consulting a third reviewer. The full text of potentially eligible studies was then retrieved and assessed for final inclusion. A total of 79 studies were finally included and incorporated into three tables categorized by target cells/ligands. These tables captured details such as study characteristics, target cell, bead properties for labeled separations, sample type, magnetic separators, flow rates/volumes tested, and efficacy and purity of the isolation. Studies lacking sufficient tabulated information were excluded from the tables but considered for qualitative synthesis if meeting inclusion criteria. Rigorous data cleaning and validation procedures were employed to maintain the precision and reliability of the final compiled dataset. This methodological approach allowed for the structured collection and presentation of evidence pertaining to target cells isolation using magnetic techniques, facilitating comparative analyses to determine optimal approaches for disease diagnosis.

3. Separation of circulating tumor cells from blood for cancer diagnosis and prognosis

3.1. Magnetophoresis of CTCs with EpCAM-based strategies

CTCs are known to be potential biomarkers for the diagnosis and prognosis of cancer. It has been demonstrated that IMNs functionalized with biomarkers that target cancer cell receptors can be used to isolate CTCs in blood samples by employing magnetic field gradients.(Beije et al., 2015; Hu et al., 2021; Li et al., 2020) Since most cancer types are of epithelial origin, the surfaces of the CTCs have a high expression of a protein known as EpCAM.(van der Toom et al., 2016) Therefore, ligands that target the EpCAM receptor are the most widely used capture agents exploited for CTC enrichment from blood samples. Several commercial magnetic separation platforms—such as Isoflux, MagSweeper, EasySep^™ EpCAM Positive Selection Kit, and the FDA-cleared CellSearch^® system—have exploited EpCAM as a target marker. (Allard et al., 2004; Harb et al., 2013; Liu et al., 2011; Talasaz et al., 2009) However, these platforms demonstrate moderate performance with respect to capture efficiency, purity, throughput, and cell viability. To overcome these limitations, numerous studies have sought to enhance these parameters using new EpCAM-targeted approaches. Table 1 reports current studies where IMNs, functionalized with biological ligands targeting the EpCAM receptor, are used for CTC isolation from blood. This section summarizes the main results from the studies reported in Table 1 investigating the immunomagnetic-based separation of CTCs in blood samples using EpCAM-based approaches.

Table 1.

Magnetophoresis of CTCs using EpCAM targeted separation.

Cancer type	Beads/particles			Sample	Magnetic separator	Batch/Continuous	Flow rate/Volume	Quantitative Separation factors	Comments	Ref
	Material	Size	Properties
Breast cancer	IMNs coated with protein corona (anti-EpCAM antibody)	250 nm; shell thickness: ~ 9 nm	Zeta potential: −8.0 mV; Magnetization: 80 emu/g	105 MCF-7 cells/mL spiked in healthy donor’s lysed blood	-	Batch	1 mL	Isolation efficiency: 90.2%; Purity: 50–100%	Successfully captured CTCs with high purity in 24/26 female breast cancer patients’ blood samples. CTCs released by trypsinization had 100% viability and proliferated for up to 3 days	(Jiang et al., 2022)
Lung cancer	Fluorescent IMNs conjugated with streptavidin (SA) (anti-EpCAM antibody)	40–95 nm	Magnetization: 6.04 – 41.6 emu/g	HCC827 and A549 cells spiked in blood	-	Batch	-	Capture efficiency from 70–95%; Viability rate: 95%	Simultaneous tumor cell fluorescent immuno-labeling and magnetic isolation. Reverse transcription polymerase chain reaction (RT-PCR) detected the 214-base pair (bp) DNA band of EGFR coding region on CTCs bound to IMNs	(P. Zhang et al., 2021)
Prostate cancer	IMNs coated with RBCs’ membranes (anti-EpCAM antibody)	80 nm; shell thickness: ~ 9 nm	-	PC-3 cells spiked in whole blood	-	Batch	1 mL	Capture efficiency: 95.71%	Minimal nonspecific adsorption. Sanger sequencing and polymerase chain reaction (PCR) detected a mutated PIK3CA gene on captured CTCs	(Meng et al., 2019)
Breast cancer and colorectal cancer	Mg particles coated with IMNs and conjugated with glutathione linker (GSH), fourth generation (G4) dendrimer, and cyanine 5 NHS (Cy5) dye (anti-EpCAM antibody)	~ 20 nm	Zeta potential: −6.51 mV	HCT116 and MCF-7 cells spiked in healthy blood	-	Batch	1 mL	Capture efficiency: > 98%	Immunocytochemistry confirmed that 0–5 CTCs per mL of blood were captured in the samples from metastatic cancer patients	(Wavhale et al., 2021)
Breast cancer	Poly(N-isopropylacrylamide)-co-Acrylic Acid microgel loaded with IMNs (anti-EpCAM antibody)	~ 670 nm	Zeta potential: −12.7 mV	MCF-7 cells spiked in blood	-	Batch	1.5 mL	Capture efficiency: 70%; Purity: 65%; Viability: >80%	3D cell culture platform was developed and tumor spheroids containing live cells were formed after 5 days of incubation	(Seyfoori et al., 2019)
Breast cancer	Carboxyl iron oxide IMNs and SA-coated polystyrene beads (anti-EpCAM antibody)	300–400 nm (iron oxide); 500 nm (polystyrene)	-	SK-BR-3 cells spiked in healthy blood samples	-	Batch	1 mL	-	Linear range of captured CTCs was 50–1000 cells/mL. Detection limit: 7 cells/mL	(Yang et al., 2019)
Metastatic carcinoma	Carbon-coated cobalt IMNs (anti-EpCAM antibody)	~ 28 nm	-	Blood samples from metastatic carcinoma patients	Commercial magnetic columns (Miltenyi Biotec)	Batch	475 μL	≥68% of CTCs captured from 6 patient samples	-	(Doswald et al., 2022)
Breast cancer and prostate cancer	SA-coated IMNs functionalized with a hydrogel (sulfobetaine methacrylate and methacrylic acid cross-linked with N,N-bis(acryloyl)cystamine) (anti-EpCAM antibody)	150 nm; hydrogel layer thickness: 6 nm	-	MCF-7 cells spiked in healthy blood samples and breast cancer and prostate cancer patient samples	-	Batch	1 mL	Capture efficiency: 96%; Recovery rate: 96%; Viability: 95%	Immunocytochemistry confirmed that 1–12 CTCs were detected in blood samples of 3 breast cancer patients and 2 prostate cancer patients. No CTCs detected in the blood of healthy donors	(Zhili Wang et al., 2021)
Head and neck cancer	Cellulose nanocrystals or nanofibers with IMNs (anti-EpCAM antibody)	15 nm	Zeta potential: −31 mV (cellulose nanocrystals); −32 mV (nanofibers)	Blood samples from head and neck cancer patients	N52-grade magnet	Batch	1.5 mL	Capture efficiency: 40% for the fibers, 72% for the crystals	The capture performance of these nanomaterials was comparable to that of the commercially available OncoDiscover® platform	(Hazra et al., 2024)
Prostate cancer	Nano-filopodia, oleic acid-capped IMNs with SA (anti-EpCAM antibody)	3.54 μm	-	Prostate cancer and benign prostatic hyperplasia (BPH) patients’ blood, and healthy blood samples	Commercial magnet (Invitrogen)	Batch	3 mL	Capture efficiency: 86.5%	CTCs were detected in 20/21 prostate cancer and 3 BPH patients’ samples blood, but no CTCs were detected in healthy blood	(Zhang et al., 2023)
Breast cancer	ZnS/Mn2+ quantum dots and SiO2 coated IMNs (anti-EpCAM antibody)	15–25 nm	Magnetization: 2.05 emu/g	Blood from breast cancer patients	-	Batch	1 mL	5–29 CTCs/mL	Simultaneous capture and detection of CTCs with a three-color immunofluorescence assay	(Cui et al., 2019)
Breast cancer	Au coated IMNs (anti-EpCAM antibody)	110 nm	Zeta potential: −63mV	MCF-7 cells spiked in lysed blood	-	Continuous	500 μL/min	Purity: ~80%	A spiral inertial microfluidic chip was employed	(Gou et al., 2021)
Liver cancer	SA-modified IMNs (anti-EpCAM antibody)	1 μm	-	HEP3B cells spiked in healthy blood samples	Rectangular, nickel-coated Nd2Fe14B magnet (0.3 T)	Continuous	0.75 mL/h	Capture efficiency: >80%; Recovery rate: 93.8%; Purity: 89.1%	Microfluidic assay with a nano-roughened channel substrate, where CTCs were trapped. No adverse effect was observed during the long-term culture of captured CTCs	(L. Zhang et al., 2019)
Colon and breast cancer	Amine-modified IMNs (monoclonal antibody CO17–1A)	50 nm	-	Caco-2 and MCF-7 cells spiked in whole blood; 1–104 cells/mL	Permanent magnet (527 mT)	Continuous	5 mL/min	Capture efficiency: 80–90%; Limit of detection: 10 CTCs/mL	3D printed immunomagnetic concentrator and adenosine triphosphate (ATP) luminescence assay to simultaneously isolate and detect CTCs in blood	(Park et al., 2021)
Breast, lung, and colon cancer	SA-coated IMNs (anti-EpCAM antibody)	-	Magnetization: 30 emu/g	MCF-7, MDA-MB-231 and SK-BR-3 cells spiked in healthy blood samples and 11 breast, lung, and colon cancer patients’ samples	NdFeB magnet, (126–252 mT)	Continuous	30 μL/min	Capture efficiency: 90%; Purity: 90%; Viability: >91%	Microdevice with a size-selective lateral flow microarray (LFM) chip and a gold-nanorod pre-embedded gelatin capture unit. Rapid dissolution of gelatin at 37°C and photothermal action of gold nanorods released CTCs. Captured CTCs had lower EpCAM expression and exhibited higher resistance to chemotherapeutic drugs	(Lv et al., 2023)
Pancreatic cancer	IMNs coated with polyethylene(glycol) (PEG) and SA (anti-EpCAM antibody)	20.3 nm	Magnetization: 85.88 Am2/kg	BxPC3 cells spiked in healthy blood samples	Two B888-2PE-N52 magnets	Continuous	2 μL/s	Capture efficiency: 60%; Purity: 34%	Antibody functionalized herringbone microfluidic capture device with a planar Halbach array	(Unni et al., 2020)
Liver cancer	IMNs functionalized with SiO2 and modified with glycidyl methacrylate (anti-EpCAM antibody)	10–20 nm	-	HepG2 cells spiked in healthy blood samples		Continuous	2 μL/min	Capture efficiency: 74–89%; Purity: 82–98%; Recovery rate: 94.1%	Lanthanide-labeled anti- asialoglycoprotein receptor (ASGPR) monoclonal antibody and anti-EpCAM-modified magnetic beads acting as a signal probe in a microdevice with a mixing and separation zone. Chip coupled with inductively coupled plasma mass spectrometry (ICP-MS) to determine the level of ASGPR on separated HepG2 cells	(Xu et al., 2023)
Breast cancer	IMNs coated with SiO2 (anti-EpCAM antibody)	5–20 nm	-	MCF-7 cells spiked in whole blood	Block of NdFeB magnets (42 MGOe)	Continuous	0.5 μL/min	Capture efficiency: 93.13%	A silicon nanowire (SiNWs) substrate in a microfluidic device to trap and isolate CTCs from whole blood	(Ghafouri & Badieirostami, 2021)
Breast cancer	IMNs (anti-EpCAM antibody)	20 nm	-	MCF-7 cells spiked in whole blood	-	Continuous	1000 μL/min	Capture efficiency: 89% and 94.5%; Purity: 84% and 92.5% (in an inertial and hybrid device, respectively)	Investigated the separation of CTCs by two microfluidic devices, including a serpentine inertial device and a hybrid inertial and magnetophoretic device	(Nasiri et al., 2022)
Breast cancer and hepati-cellular cancer	IMNs modified with polyethyleneimine (PEI) and carboxylated graphene nanosheets, coated with leukocyte membranes (anti-EpCAM antibody)	16.94 nm	Magnetization: 57.2 emu/g; Zeta potential: −28.7 mV	MCF-7 cells and HepG2 cells spiked in whole blood	-	Batch	1 mL	Capture efficiency: 88.9%; Purity: 94.4–98.2%; Viability: 98%	2–48 CTCs detected in 8 cancer patient samples. Superior capture efficiency and purity compared to commercial MACS beads	(Zhou et al., 2019)
Breast cancer	IMNs coated with J774A.1 leukocyte membrane fraction (PEG 2000-distearoylphosphatidylethanolamine (DSPE-PEG2000) modified SYL3C aptamer)	~ 80 nm	Magnetization: 80 emu/g	MCF-7, 4T1, or MDA-MB-231 cells spiked in whole blood	Permanent magnet	Continuous	1 μL/min	Capture efficiency: > 91%	The prepared IMNs were arranged in a nickel square of a microfluidic device to improve local topographic interactions with CTCs	(F. Zhang et al., 2019)
Breast cancer	SA conjugated IMNs (EpCAM aptamer)	1 μm	-	MCF-7 cells spiked in blood	N52 magnet	Continuous	2 μL/min	Capture efficiency: 89%; Recovery rate: 84%; Viability: 99.3%	Cascaded phase transfer microfluidic chip with three regions (sorting, purification, and release zone)	(Nian et al., 2024)
Breast cancer	IMNs encapsulated with neutrophil membranes (anti-EpCAM antibody)	-	-	MCF-7 cells spiked in whole blood	-	Batch	1 mL	Capture efficiency: 96.82%; Purity: 90.68%	CTCs were isolated in 19/20 blood samples from breast cancer patients. PCR and Sanger sequencing detected the 3140A/G mutation on the isolated CTCs	(Xianjia Wu et al., 2022)
Breast cancer	IMNs coated with SiO2-NH2 (SYL3C aptamer-mediated DN concatemer (AMDC))	500 nm; SiO2 shell thickness: 50 nm	Zeta potential: −27.54 mV	MCF-7 cells spiked in whole blood	-	Batch	400 μL	Capture efficiency: 66%; Viability: 92%	Stimulus responsive aptamers were used for both capturing and releasing CTCs with little influence on cellular activity	(Zhang et al., 2022)
Breast cancer	Magnetic graphene oxide coated with MCF-7 tumor membrane (Multivalent SYL3C aptamers)	-	-	MCF-7 cells spiked in healthy blood samples	-	Batch	1 mL of blood	Capture efficiency: 79%; Purity: 83%; Viability: 98.3%	Detection limit of 5 cells/mL in blood	(Jia et al., 2023)

The effectiveness and specificity of the capture procedure are crucial parameters for the quick and precise CTC isolation for early diagnosis or tracking the course of malignancy. Prior research has utilized commercial IMNs coated with mono- and polysaccharides for the magnetophoretic capture of tumor cells.(Issadore et al., 2012; Scarberry et al., 2011) However, these traditional methods have demonstrated low specificity since biomolecules in the blood nonspecifically adsorb onto the surface of the particles, resulting in the in-situ formation of a protein corona that covers the surface ligands of the IMNs, reducing their targeting efficacy.(Scarberry et al., 2011) On the contrary, coating the IMNs with polymers, such as polyethylene glycol (PEG), is a classical method employed to minimize non-specific interactions. For example, Unni et al. investigated the capture of CTCs by PEG-coated, anti-EpCAM functionalized IMNs in a herringbone microfluidic device with a glass substrate functionalized with anti-EpCAM antibodies.(Unni et al., 2020) The tailored nanoparticles showed minimal non-specific adsorption to CCRF-CEM leukemia cells with a low expression of EpCAM but a strong affinity for BXPC3 pancreatic tumor cells with a high expression of EpCAM. In addition, this work demonstrated that coupling magnetic forces with an antibody-mediated microfluidic surface capture led to markedly improved capture efficiency and specificity in the presence of a strong magnetic field gradient generated by a Halbach array. The capture efficiency was approximately 60% for blood samples spiked with BXPC3 cells injected into the microfluidic device at flow rates as high as 2 μL/s (7.2 mL/h). However, the purity of the captured cells was 34% for the spiked blood samples compared to nearly 100% for the cell mixtures in PBS, which was attributed to the white blood cells (WBCs) present in the blood sample, having a greater binding affinity to the surface of the microfluidic device.

However, there have been validity and safety concerns about coating IMNs with polymers. Thus, researchers have focused on coating the particles’ surface with cellular membranes, otherwise known as biomimetic nanoparticles, to reduce nonspecific biomolecule adsorption and uphold the materials’ targeting efficacy in complex physiological environments.(Meng et al., 2019) Another issue that can be overcome with this approach is the low purity of enriched CTC samples, which poses substantial obstacles to downstream analysis of these cells. This problem results from the significant amount of nonspecifically bound leukocytes that disrupt the separation platform, making it more difficult to conduct follow-up analyses and investigations accurately and consistently; this analysis can offer vital information about cancer prognosis, metastasis, and therapeutic effectiveness.(Pipatwatcharadate et al., 2023) (Meng et al., 2019)

Utilizing the biomimetic nanoparticle approach, Meng et al. developed IMNs coated with RBC-membrane-derived vesicles and functionalized with anti-EpCAM antibodies that effectively isolated CTCs from clinical blood samples.(Meng et al., 2019) After incubating IMNs and RBC-IMNs in human plasma, there was a noticeable increase in the size of the IMN brought on by the formation of protein coronas on IMNs. On the other hand, there was no discernible alteration in the size of RBC-IMNs, indicating that the RBC coating successfully blocked the nonspecific biomolecule adsorption. As seen in Figures 3(A) and (B), RBC-IMNs maintained their structure after being mixed with PC-3 cells, while IMNs were internalized into the cells due to the formation of a protein corona. Inductively coupled plasma-atomic emission spectrometry (ICP-AES) determined that interactions between RBC-IMNs and PC-3 cells were enhanced (Figure 3(C)) and were not time-dependent (Figure 3(D)). Figure 1(E) shows that IMNs and RBC-IMNs demonstrated excellent capture (~90%) of EpCAM-positive PC-3, HeLa, and MCF-7 cancer cells in PBS. The cell separation efficiency of IMNs and RBCs-IMNs was 60.22% and 95.71% in the spiked blood samples, respectively, as shown in Figure 3(F). The decrease in the capture efficiency of IMNs was attributed to the formation of the protein corona around the IMNs in the blood sample. Additionally, RBC-IMNs could isolate CTCs from 28 out of 30 blood samples from prostate cancer patients with a higher purity than IMNs. This study successfully demonstrated that these RBC-IMNs inhibited nonspecific adsorption in complex bodily fluids while retaining their capacity to target CTCs.

Figure 3.

Scanning electron microscopy (SEM) images of a PC-3 cell captured by (A) IMNs or (B) RBC-IMNs. Scale bars are 1 μm and 500 nm for the left and right panels, respectively. Interactions between PC-3 cells and the different types of IMNs, (C) after 24-hour incubation at various concentrations and (D) after various incubation times at an IMN concentration of 100 μg per mL, determined by ICP-AES. (E) Cell capture efficiency of different types of IMNs in PBS. (F) The capture efficiencies of the different IMNs to PC-3 cells in mimic clinical samples. Reproduced with permission,(Meng et al., 2019) under a Creative Commons license CC BY 4.0.

In another study, Zhou et al. created leukocyte-mimicking biomimetic immuno-magnetic nanospheres (BIMNs) combining graphene nanosheets and IMNs to target EpCAM-positive CTCs.(Zhou et al., 2019) The remarkable sensitivity of this sophisticated material was highlighted by its detection limit, which was as low as 3 CTCs in 1 mL of blood. The capture efficiency and purity of the CTCs isolated by BIMNs were above 85.0% and 94.4% in 1 mL blood samples spiked with 20–200 MCF-7 cells after a 2-minute incubation. Notably, 98.0% of the isolated MCF-7 cells were viable and could proliferate in vitro for many passages. This high viability highlighted the delicate nature of the capture procedure, which is essential for biomedical studies. Additionally, the validity of BIMNs was confirmed using peripheral blood samples from cancer patients, and good consistency was shown in the counting of CTCs, with a relative standard deviation (RSD) of 8.7 ± 5.6%.

Utilizing biomimetic nanoparticles has several benefits, including high capture efficiency and improved CTC purity, making them a valuable tool for isolating CTCs.(Zhang & Wang, 2024) However, conventional systems based on biomimetic nanoparticle-based separation approaches have some limitations.(F. Zhang et al., 2019) To overcome these issues, biomimetic nanoparticles coupled to microfluidics have been presented as an alternative to efficiently capture and detect CTCs. For instance, Zhang et al. developed a nickel-pattern microfluidic device loaded with leukocyte membrane-coated IMNs functionalized with PEG₂₀₀₀-distearoylphosphatidylethanolamine (DSPE-PEG2000) modified SYL3C aptamers (A-M/NCs) to capture CTCs, as shown in Figure 4(A).(F. Zhang et al., 2019) Figure 4(B) exhibits the microfluidic channel with the uniform nickel pattern fabricated on the bottom of the microchannel, with the help of permanent magnets that improve the capturing capabilities of the CTCs. This device could capture more than 91% of CTCs (MCF-7, 4T1, or MDA-MB-231) spiked in whole blood samples within 20 minutes with minimal nonspecific adsorption, as shown in Figure 4(C).

Figure 4.

(A) Schematic of A-M/NCs and tumor cell capture within a microfluidic system. (B) Schematic of nickel patterns fabricated on the bottom of the capillary microchannel. (C) The capture efficiency of various types of cells using this biomimetic microfluidic system. Reproduced with permission,(F. Zhang et al., 2019) under a Creative Commons license CC BY 4.0.

Several studies have reported that IMNs have low stability in biological fluids and modify the cell’s biological functions after nonspecific endocytosis.(Saeed et al., 2015; Xu et al., 2011) One way to overcome this challenge is to encapsulate IMNs in a biocompatible polymeric matrix, otherwise known as a nanohybrid microgel. For example, Seyfoori et al. developed a soft, thermo-pH-responsive microgel encapsulated with superparamagnetic iron oxide nanoparticles for isolating MCF-7 breast cancer cells.(Seyfoori et al., 2019) To create the nanohybrid microgel, emulsion polymerization was carried out, resulting in an anionic poly(N-isopropylacrylamide)-co-acrylic acid (PNIPAM-AA) polymeric matrix, and then IMNs were precipitated in situ within the structure. Anti-EpCAM antibodies were conjugated to the carboxyl groups of the polymeric matrix with and without a protein G linker. Protein G binds to the fragment-crystallizable (Fc) region of the antibody so that the paratopes of the antibody are oriented to face the epitopes of the antigens. In blood samples spiked with 4×10² MCF-7 cells, the maximum capture efficiency for the oriented antibody-conjugated microgel was 80%, compared to 72.1% from the non-oriented antibody-conjugated microgel. The purity of the CTCs isolated by the oriented antibody-conjugated microgel was 60% in a spiked blood sample. Furthermore, no appreciable nonspecific cell capture (C2C12 and U-87 cells) was observed with these microgels.

After being isolated, CTCs should ideally be released for further downstream clinical analysis. If the cells are viable after release, the IMNs can be considered biocompatible and non-toxic. Then, gene analysis can be carried out on the CTCs to determine the underlying mechanisms of metastasis, tumor heterogeneity, and genes contributing to drug resistance or disease relapse, which can provide a foundation for personalized medicine.(Lu et al., 2020) In the study by Meng et al., 80 nm RBC membrane-coated and anti-EpCAM antibody-functionalized IMNs were utilized to capture PC-3 cancer cells. Gene mutation analysis was then carried out on these isolated CTCs using polymerase chain reaction and Sanger sequencing.(Meng et al., 2019) The CTCs were determined to have a positive expression of the 3140A/G mutation, which was associated with cancer metastasis, and WBCs did not display this mutation. 3D cell culture can also be carried out on the CTCs to determine their ability to form spheroids. As shown in Figures 5(D) to 5(F), Seyfoori et al., investigated spheroid formation with a 3D microwell array, after capturing MCF-7 cells with an anionic IMN-loaded PNIPAM-AA microgel functionalized to anti-EpCAM antibodies. Following five days of incubation, the majority of the CTCs were viable and successfully proliferated (Figures 5(A) to 5(C)), with a few solitary cells giving rise to microtumor spheroids with a dimension of 100 μm (Figures 5(G) to 5(L)). 3D cell culturing to determine spheroid formation can be used to determine the response of CTCs to chemotherapeutic drugs.(Seyfoori et al., 2019)

Figure 5.

(A−C) 2D proliferation of the magnetically separated CTCs in a 96-well plate over 7 days. (D-E) Illustration of the designed and (F) 3D-printed agarose microwell for cell aggregate formation. (G-J). Spheroid formation after 7 days of CTC incubation in the microwell array. Fluorescent images of the spheroids incubated with a (K) Live/Dead staining kit and (L) 4′,6-diamidino-2-phenylindole (DAPI) nuclei staining. Reproduced with permission,(Seyfoori et al., 2019) under a Creative Commons license CC BY 4.0.

3.2. Magnetophoresis of CTCs with non-EpCAM-based strategies

As presented previously, a vast majority of the existing methods for isolating CTCs rely on IMNs functionalized with aptamers or antibodies that specifically target the EpCAM receptor on the cells surface, which might not be suitable for the isolation and purification of all the CTCs in the sample. The disadvantages of EpCAM-targeted separations include the inability to isolate cells that have undergone epithelial-to-mesenchymal transition and the non-specific adsorption of background leukocytes and EpCAM-positive circulating epithelial cells onto the IMNs. For example, it has been suggested that skin cells containing epithelial biomarkers may contaminate the blood samples when the needle penetrates the skin of cancer patients.(Hyun & Jung, 2014) Thus, EpCAM-based approaches for CTC isolation have recently been replaced or combined with systems that can capture CTCs with a low expression of EpCAM. With these systems, the separation efficiency can be enhanced by using IMNs labeled with multiple antibodies targeting various antigens that can capture different subpopulations of CTCs. Table 2 presents the recent developments in the magnetic isolation of CTCs via non-EpCAM-based (or hybrid) enrichment strategies.

Table 2.

Magnetophoresis of CTCs using non-EpCAM targeted separation.

Cancer type	Beads/particles				Sample	Magnetic separator	Batch/Continuous	Flow rate/Volume	Quantitative Separation factors	Comments	Ref
	Material	Size	Targeting ligands	Properties
Breast cancer	IMNs coated with MCF-7 cancer cell membranes (CMs) and leukocyte membranes (LMs)	Diameter: ~100 nm	Charge-based separation (Label-free)	Magnetization: 59 emu/g	MCF-7 cells (105) spiked in lysed blood	NdFeB permanent magnet (0.2 T)	Batch	5 mL	Capture efficiency: 72.4%; Recovery rate: 85.1%; Purity: 91.2%	Higher separation purity and better detection rate in orthotopic 4T1 mouse metastatic mammary carcinoma models than the anti-EpCAM antibody-conjugated IMNs	(Chang et al., 2020)
Breast cancer	IMNs modified with tetraethyl orthosilicate (TEOS), 3-mercaptopropyltrimethoxysilane (MPTMS) and 2,2’-dithiodipyridine (DDP)	Diameter: 261.1 nm	Hyaluronic acid (HA)- functionalized L-cysteine ethyl ester hydrochloride (Cys)	Zeta potential: −29 mV; Magnetization: 39.75 emu/g	MCF-7 cells spiked in whole blood	-	Batch	1 mL	Capture efficiency: 66.6%−80%; Release efficiency: 81.4%; Limit of detection: 150 cells in 1 mL	Almost all released CTCs were viable, as determined by fluorescence microscopy. Released CTCs proliferated without changes in morphology and behavior	(Y. Zhang et al., 2021)
Ovarian cancer	IMNs coated with PEI and CdSe/ZnS quantum dots (QDs)	Diameter: 400 nm	Folic acid (FA) functionalized with NH2 and PEG (NH2-PEG5000-FA)	-	Whole blood from ovarian cancer patients	-	Batch	3 mL	Capture efficiency: >85%; Viability: >90%	2–12 CTCs were detected in the samples of 10 cancer patients	(Pan et al., 2022)
Colon cancer	carboxyl-modified IMNs	Diameter: 1.02 μm	Ulex europaeus agglutinin-I (UEA-I)	-	Hoechst 33342-prestained SW480 cells spiked in blood	-	Batch	1 mL	Capture efficiency: 89%; Viability: 93%	3 to 20 CTCs/mL were captured from samples of cancer patients	(Tian et al., 2022)
Colorectal, lung, breast, prostate cancer	Commercial Sera-MAG™ SpeedBeads SA-blocked magnetic particles (GE Healthcare)	Diameter: 1 μm	Two recombinant versions of VAR2CSA protein	-	COLO205, A549, SW480, SK-BR-3 and PC-3 cells spiked in healthy, lysed blood	-	Batch	3 mL	Capture efficiency: 12.3–69.4% and 47.2–111.1% for direct and indirect capture, respectively	Two types of capture assays were performed: direct and indirect	(Sand et al., 2020)
Ovarian, lung, cervical cancer	Halloysite-nanotubes coated with B-cyclodextrin	External diameter, internal diameter, length, and wall thickness of 40–60 nm, 15–20 nm, 50–1,000 nm and 22–25 nm, respectively	FA	Magnetization: 20 emu/g	Skov3, Hela or A549 cells spiked in rabbit blood samples	-	Batch	-	Capture efficiency: 93%	The reusability of the nanomaterials was investigated, where the capture efficiencies were >90% for the three cancer cell lines after three regeneration cycles	(Li et al., 2019)
Ovarian cancer	SA-coated IMNs	Diameter: 70.4 nm	FA-functionalized Poly(amidoamine) (PAMAM) dendrimer	Zeta potentials of IMNs and FA-PAMAM-IMNs of −46.32 and −6.3 mV, respectively	SKOV-3 cells spiked in healthy blood samples	-	Batch	1 mL	Capture efficiency: 79.6%	The number of CTCs detected in 9 ovarian cancer patient samples ranged from 2–11, and no CTCS were detected in 11 healthy donor samples	(Meng et al., 2020)
Breast, kidney, prostate, lung, ovarian, colon, bronchial, esophageal carcinomas	IMNs	Diameter: 147.9 nm	Tannic acid	Zeta potential: −33.9 mV	Blood from 6 healthy donors and 21 cancer patients	-	Batch	1 mL	Capture efficiency: 62.3–93.7%; Purity: 94.5%; Viability: 95–97%	1–10 CTCs detected in the samples of different cancer patients and no CTCs detected in the healthy samples	(Ding et al., 2021)
Acute lymphoblastic leukemia	SA conjugated IMNs	Hydrodynamic diameter: 1,696.4 nm	Aptamer Sgc8		CCRF-CEM (human T-cell leukemia) cells spiked in healthy blood samples	-	Batch	1 mL	Capture efficiency: 80–84%; Limit of detection: 26 cells/mL	Long ssDNA molecules with hundreds of repeating aptamer units supported on IMNs via rolling cycle amplification	(Lv et al., 2021)
Head and neck cancer	Modified cellulose nanocages with Fe3O4	Diameter: 80–140 nm	Tf	Zeta potential: +21 mV at pH=5, −22 mV at pH=7	Blood samples from head and neck cancer patients	-	Batch	5 mL	Capture efficiency: 85%	This system had a lower capture efficiency than the commercial Oncoviu kit	(Hazra et al., 2020)
Breast cancer	γ-Fe2O3 nanorods/carbon dots nanomotors	Width: 80–100 nm; Length: 350–450 nm; Diameter (QDs): 3–5 nm	FA and HA	-	Blood samples from tumor-bearing female mice	-	Batch	-	Capture efficiency: 97.5%	Propulsion from the magnetic field and H2O2 solutions overcame the Brownian motion resulting in a fast capture of CTCs	(Ren et al., 2023)
Lung, liver cancer	Au-IMNs	-	Aptamers (p-A549 and p-SMMC)	-	A549 and SMMC-77221 cells spiked in healthy serum samples	-	Batch	50 μL	Capture efficiency: 93–108%	Indirectly determined the number of captured CTCs using ICP-MS	(Ye He et al., 2019)
Acute lymphoblastic leukemia	SA-coated IMNs	350 nm	Aptamer sgc8c	-	Blood samples from leukemia patients and healthy donors (RBC lysis and WBC collected)	-	Batch	1 mL	Capture efficiency: 97.81–102.71%; CTCs detected in a linear range of 200–10,000 cells/mL with a detection limit of 100 cells/mL	Luminol- H2O2 system lysed the captured CTCs, and the increased production of singlet oxygen increased the chemiluminescence signal	(L. Ding et al., 2020)
Lymphoma and Burkitt lymphoma	Magnetic graphene nanoribbons with boronic acid	11 nm	Anti-CD20 antibody	-	Whole blood from two cancer patients	-	Batch		Limit of detection: 38 cells/mL; Linear detection range: 100–1,000,000 cells/mL	No interference with MCF-7 (breast cancer), HEK293 (human embryonic kidney), and HL-60 and KCL-22 (leukemia) cells. After separation, cells were placed in an electrochemical cell to record the impedance	(Hashemi et al., 2020)
Acute lymphoblastic leukemia	Platinum and iron oxide-coated TiO2 nanotubes	Nanotube diameter: 300 nm; Nanotube length: 7 μm (Fe2O3: 10 nm)	Aptamer sgc8c	Zeta potential: −33.1 mV	CCRF-CEM cells spiked in healthy blood samples	Permanent magnet (0.1 T)	Batch	1 mL	Capture efficiency: 66–80%	A rotating magnetic field was used. Captured cells inactivated with visible-light irradiation due to reactive oxygen species generation from the photocatalytic TiO2	(Zhao et al., 2021)
Acute lymphoblastic leukemia	IMNs with self-assembled aptamer/protein probes	250 nm	Aptamer sgc8c	Zeta potential: −11.1 mV	CCRF-CEM cells spiked in healthy blood samples (RBC lysis and WBC collected)	-	Batch	-	Detection limit: 200 cells/mL; Linear detection range: 150–10,000 cells/mL	Aptamer-modified IMNs for CTC capture and self-assembled aptamer/protein hybrids for signal amplification. Tween-20 was added to reduce nonspecific adsorption	(Ding et al., 2019)
Breast cancer	SiO2 coated IMNs, PEI modified- or carboxylated (IO-PEI or IO-COOH), or carboxylated IMNs, PEI modified-graphene oxide nanosheets (IO-rGO)	100 nm	Charge-based separation (Label-free)	Zeta potential: −14 mV; Magnetization: 40–50 emu/g	MDA-MB-231 cells spiked in healthy blood samples after RBC lysis	-	Batch	1 mL	Capture efficiency: 63–69%; Purity: 63–69%	The IO-rGO nanocomposites had higher cytotoxicity to the CTCs than IO-PEI or IO-COOH spherical particles due to nutrient depletion and destruction of pseudopods	(Yishu He et al., 2019)
Breast cancer	Carboxymethyl dextran coated IMNs	25 nm	Charge-based separation (Label-free)	Zeta potential: −56 mV; Magnetization: 73 emu/g	25 blood samples from breast cancer patients	-	Batch	2 mL	Capture efficiency: 86% (24 hour stored blood samples), 97% (fresh blood samples); Viability: 56% (leukocytes in fresh samples)	The blood bag connected to a magnet acted as a separation column for the non-specific capture of tumor cells and removal of WBCs under a low magnetic field gradient	(Payer et al., 2020)
Breast cancer	IMNs coated with CaCO3 and PEI	110 nm (shell thickness: 10 nm)	Charge-based separation (Label-free)	Zeta potential: +35 mV; Magnetization: 36 emu/g	Green fluorescence protein labeled MDA-MB-231GFP cells spiked in lysed blood	-	Batch	1 mL	3–10 CTCs/mL captured in 10 clinical samples	pH-sensitive core-shell inorganic nanocomposite to isolate and subsequently release the CTCs in slightly acidic media	(P. Wang et al., 2021)
Breast cancer	IMNs coated with zeolitic imidazolate framework-8 (ZIF-8)	230 nm	Aptamer SYL3C	Zeta potential: −23.77 mV	MCF-7 cells spiked in whole blood	-	Batch	400 μL	Capture efficiency: >60%; Recovery rate: 30–42%; Viability: 96%	Integrated high-efficiency capture with simultaneous pH- and near-infrared (NIR)-irradiation to release the CTCs	(Wang et al., 2023)
Breast cancer	IMNs coated with SiO2 and a hybrid membrane modified with SA	Hydrodynamic size: 191.3 nm	Aptamer Tetra-DNA-Ag2S	Zeta potential: −29.5 mV	MCF-7 cells spiked in lysed blood and whole blood	-	Batch	1 mL	Capture efficiency: 90.25% (whole blood), 96.24% (lysed blood); Detected 6–10 cells per L in blood samples of 8 cancer patients	Synergistic effect from multivalent aptamer conjugation and membrane coating enhanced the capture efficiency and lowered non-specific adsorption	(C. Ding et al., 2020)
Breast cancer	IMNs coated with (3-aminopropyl) trimethoxysilane (APTES)	39.14 nm	Anti-HER2 antibody	Magnetization: 64.5 emu/g	SK-BR3 cells spiked in whole blood	-	Batch	1 mL	Capture efficiency: 77–98%	Limit of detection: 100 CTCs in more than 6 million RBCs and WBCs	(Haghighi et al., 2019)
Prostate, breast, non-small cell and small cell lung cancer, canine melanoma cancer	Water-based ferrofluids	Diameter: 10.91 nm	Antibodies that target leukocytes	Label-free	PC-3, MCF-7, MDA-MB-231, HCC- 1806, H-1299, H-3122, DMS-79, H-69 cells spiked in WBCs (1–100 cells/mL)	Neodymium permanent magnet (1.33 T)	Continuous	100 μL/min	Capture efficiency: 94.99–99.68%; WBC depletion rate: >99%	Integrated ferrohydrodynamic cell separation for the label-free enrichment of CTCs independent of size and tumor antigen expression via the immunomagnetic capture of WBCs	(Yang Liu et al., 2021)
Breast cancer	Popcorn-shaped, core-shell iron oxide−Au IMNs	~ 100 nm	EpCAM, HER2, D44, IGF1R antibody	Zeta potential: +35 mV	K-BR-3, MDA-MB-231 or MCF-7 cells spiked in whole blood	-	Continuous	10–200 μL/min	Capture efficiencies decreased from 90 to 82% with increasing flow rate	Employed surface-enhanced Raman scattering (SERS) nanotags in a microfluidic device	(Wilson et al., 2020)
Gastric cancer	IMNs	-	Anti-CD45 antibody	-	AGS cells spiked in blood	Halbach array magnet	Continuous	6 mL/h	Capture efficiency: 96.6%; WBC depletion rate: 99.6%; 2–8 CTCs detected in 10 cancer patients	Microfluidic system for the immunomagnetic capture of WBCs and the label-free separation of CTCs	(Xue et al., 2019)
Prostate cancer	SA-coated IMNs	2.8 μm	Anti-prostate-specific membrane antigen (PSMA) antibody	-	LNCaP cells spiked in blood samples from healthy rats	Permalloy (80%Ni-20%Fe)	Continuous	200 μL/min	Capture efficiency: 70%	Integration of multiorifice flow fractionation for size-based enrichment (removal of free IMNs and RBCs) and a magnetic chip for CTC isolation	(Esmaeilsabzali et al., 2019)
Liver cancer	Iron-oxide doped biosilica frustules	17 nm	Aptamer TLSS11a	-	HEPG2 cells spiked in healthy blood samples	N45 neodynium rod and cubic magnets	Continuous	1300 μL/h	Capture efficiency: 94.6%; Purity: 89.7%	A chamber for the viscoelastic separation of RBCs subsequently followed by the magnetic isolation of CTCs from WBCs	(Mohammadi et al., 2022)
Breast cancer	SA-modified IMNs	1 μm	Anti-CD24 antibody	Zeta potential: −25 mV (pH 6), −53 mV (pH 10)	MDA-MB-231 and MCF-7 cells spiked in healthy whole blood and plasma samples	Cylindrical nickel-coated neodymium magnets (0.03 T/mm gradient)	Continuous	0.25 mm/s	Capture efficiency: 70%; Purity: >99%; Viability: 95%	A stationary oil-water interface was used. Fast assay time with good performance (15–20 minutes)	(Pirozzi et al., 2019)
Breast, non-small cell and small cell lung cancer, prostate cancer	IMNs	10.91 nm	Label-free	Magnetization: 0.104 kA/m	HCC1806, HCC70, MCF7, MDAMB-231, H1299, H3122, DMS79, H69 and PC-3 cells spiked in healthy blood samples	Four NdFeB permanent magnets (0.5–1.5 T, 625 T/m gradient)	Continuous	12 mL/h	Capture efficiency: 99.08%; WBC contamination: 533 cells per mL of blood	Integrated ferrohydrodynamic cell separation for the label-free enrichment of CTCs independent of size and tumor antigen expression via the immunomagnetic capture of WBCs	(Zhao et al., 2019)
Lung cancer	Iron-oxide clusters	180 nm	Arginyl-glycyl-aspartic acid (RGD) peptide or avidin	Zeta potential: −36.31 mV; Magnetization: 87 emu/g	A549 cells spiked in healthy blood samples	Two neodymium magnets	Batch	-	-	In the magnetic spinner, blood was mixed with the functionalized IMNs and injected into the device, that was then subjected to rotation to capture the cells. Unwanted cells were eluted, and the magnets removed to later release the CTCs	(Lee et al., 2022)
Gastric cancer	SA-modified Fe3O4 IMNs	155 nm	Aptamers EpCAM and PTK7	Zeta potential: −21.32 mV	Blood from patients diagnosed with gastric cancer	-	Batch	1 mL	Capture efficiency: >96% for MGC-803 and BGC-823 cells, 4% for THP-1 cells	PCR analysis determined that MGC-803 CTCs exhibited higher levels of multidrug resistance proteins so these CTCs are less sensitive to chemotherapeutic drugs	(Li et al., 2022)
Head and neck squamous cell carcinoma	IMNs	~ 1 μm	Anti-human EpCAM (CD326) or anti-human cell surface vimentin (CSV) antibody	-	SCL-1 or patient-derived squamous cell carcinoma spiked in whole blood	-	Batch	7.5 mL	Capture efficiency: 95%	Tumor cell isolation in a semi-automated process combined with a reduction of WBCs	(Gribko et al., 2021)
Colorectal cancer	IMNs nanoparticles coated with lipid bilayers	198.3 nm	Anti-Kirsten rat sarcoma viral oncogene (anti-KRAS) and anti-EpCAM antibodies	Zeta potential: +29.7 mV	Blood from patients with colorectal cancer	-	Batch	7.5 mL	Capture efficiency: 92.9%	Anti-KRAS antibody-modified lipid IMNs can be used in the diagnosis and treatment of KRAS colorectal cancer	(Feng et al., 2020)
Breast cancer	Hydrophobic IMNs	250 nm	Anti-EpCAM- dimethyl octadecyl epoxypropyl ammonium chloride (GHDC), anti-epidermal growth factor receptor (EGFR)-GHDC, Anti-HER2-GHDC and Anti-Mucin 1 (MUC1)-GHDC antibodies	Magnetization: 29.9 emu/g; Zeta potential: +20 mV	Whole blood from multi-tumor patients	-	Batch	7.5 mL	Capture efficiency: 87.5%	Exclusive incubation with EpCAM, EGFR, HER2 and MUC-1 targeting IMNs could achieve a higher CTC separation rate than a combination of IMNs	(Chen et al., 2019)
Breast and lung cancer	Commercial SA coated IMNs (Bangs, Invitrogen)	Diameter in the μm range	Folate and anti-EpCAM antibody	-	KB and MDA-MB-231 cells spiked in healthy blood and samples from lung cancer patients	-	Batch	~200 μL	Capture efficiency: 90%	8–84 CTCs per 8 mL of sample were found in the blood of lung cancer patients	(Y. Hu et al., 2022)
Breast cancer	SA coated, zirconium-based metal organic framework UIO-67, core-shell Fe3O4	231.91 nm	Anti-EpCAM and anti-N-cadherin antibodies	Zeta potential: −20 mV	MCF-7 and HeLa cells spiked in healthy blood samples	-	Batch	5 mL	Capture efficiency: 70% for HeLa, 75% for MCF-7	Detected between 1–10 CTCs in the 7 peripheral blood samples of patients and no CTCs were detected in any of the healthy control blood samples	(M. Hu et al., 2022)
Gastric carcinoma, liver tumor, epithelioid carcinoma	Immunolipid IMNs	Hydrodynamic size: 236.6–251.1 nm	Anti-EpCAM and anti-vimentin antibodies	Zeta potentials: + 30.8 and + 30.5 mV for anti-EpCAM-and anti-vimentin IMNs, respectively	Arterial and venous blood from patients who had surgery and healthy donors (control)	-	Batch	7.5 mL	Capture efficiency: 85–90%	The amount of CTCs captured in arterial blood was higher than that in venous blood in cases of single and multiple metastases. Next-generation sequencing showed heterogeneity among different tumor samples	(Yan Liu et al., 2021)
Prostate cancer	Gelatin and SA coated IMNs		Anti-EpCAM and anti-CD146 antibodies	-	LNCaP, PC-3, MB-231, and MCF-7 cells spiked in healthy blood samples	-	Continuous	300 μL/min	Capture efficiency: 91%; Purity: 36%	3–14 CTCs per mL of blood were detected in the samples of 10 prostate cancer patients, and no CTCs were detected in the samples of healthy donors	(Li et al., 2024)
Breast cancer	PEI-functionalized poly(styrene/acrylamide) nanospheres assembled with nano γ-Fe2O3 and quantum dots	358 nm	Anti-EpCAM, anti-EGFR and anti-HER2 antibodies	Magnetization: 14.1 emu/g	MCF-7 cells spiked in healthy blood samples	-	Batch	1 mL	Capture efficiency: >80%	Captured CTCs in the range of 3–23 CTCs per mL of blood from 10 breast cancer patients and no CTCs in the healthy samples	(Wu et al., 2020)
Colorectal Adenocarcinoma and Renal Cell Carcinoma	Magnetic levitation: CTCs levitated above WBCs, were captured, and collected	-	-	-	Blood from patient samples; after RBC lysis, CTCs were suspended in a 30 mM paramagnetic medium	Two neodymium (NdFeB) permanent magnets	Batch	-	-	Developed “Fastcount”, a MATLAB-based algorithm for precise, automated quantification and phenotypic characterization of CTCs	(Ogut et al., 2023)

The major challenge in capturing CTCs is the absence of a universal marker on the surface of CTCs that can be targeted, as CTCs have a very heterogeneous phenotypic profile, and the surface antigen expression on CTCs can be altered after cells undergo a process known as an epithelial-mesenchymal transition.(Hu et al., 2021; van der Toom et al., 2016) So, the number of captured CTCs of any system that uses only anti-EpCAM antibodies may be underestimated since EpCAM-negative cells are not captured. In order to enhance the isolation process, researchers have focused on various approaches, including multiplexed techniques to capture and detect CTCs to reduce false negatives and improve capture accuracy. With this approach, IMNs can be functionalized with biomarkers that target multiple cancer-specific receptors. In this section, the most current, novel systems using non-EpCAM targeting separation platforms are presented.

As introduced in Table 2, multiple surface receptors different from EpCAM have been pursued in the last few years for CTC enrichment. For example, Hashemi et al. introduced a novel method for signal amplification to identify lymphoma cancer cells by using magnetic graphene nanoribbons (MGNRs) functionalized with anti-CD20 antibodies. CD20 is a B-cell-specific, nonglycosylated phosphoprotein expressed in more than 90% of B-cell lymphomas.(Hashemi et al., 2020) For oriented antibody immobilization, the MGNRs were coated with boronic acid before antibody incubation because it can interact with the Fc region of the antibody without the need for spacers. After the CTCs were captured by the anti-CD20 functionalized MGNRs, the immunocomplex was magnetically collected on a screen-printed carbon electrode in an electrochemical cell to measure impedimetric signals. This study detected CTCs in the linear range of 100−1,000,000 cells/mL with a detection limit of 38 cells/mL.

Exploiting multiplex detection, Wilson et al. introduced a method for isolating CTCs by integrating microchip-based immunomagnetic isolation with magnetic surface-enhanced Raman scattering (SERS) nanotags for multicolor detection.(Wilson et al., 2020) Anisotropic iron oxide-gold, core-shell nanoparticles were functionalized with SERS nanotags (anti-EpCAM, anti-HER2, anti-CD44, anti-insulin-like growth factor 1 receptor (IGF1R) antibodies were combined with 4 different Raman receptors) to capture the cancer cells, which were subsequently detected by SERS within a microfluidic device. Thus, this system could perform SERS detection, optical imaging, and cell isolation within one microfluidic device. Therefore, this multiplexed method offered various benefits, reduced the number of false negative results, and facilitated automation, unlike conventional technologies based on single-cell analysis.

In a novel work carried out by Wu et al., MCF-7 cells were captured in a multifunctional nanosphere-mediated microfluidic platform integrated with a single-cell fluorescence detection platform, a significant advancement in the field of cancer study.(Wu et al., 2020) In this platform, anti-EpCAM, anti-EGFR, and anti-HER2 antibodies were functionalized on optically encoded magnetic beads (OEMBNs), which were an assembly of PEI-functionalized poly(styrene/acrylamide) nanospheres, γ-Fe₂O₃ nanoparticles, and quantum dots. As shown in Figure 6(A), over 80% of MCF-7 cells spiked into 1 mL of whole blood were captured with this novel material, at concentrations ranging from 15–1,000 cells per mL. Immunohistochemistry further demonstrated the capture of CTCs by the OEMBNs (Figure 6(B)). In addition, the OEMBNs captured CTCs in the blood samples of 10 breast cancer patients in the range of 3–23 CTCs per mL and did not capture any CTCs in 3 healthy blood samples, as shown in Figure 6(C). Overall, this platform was able to capture over 91% of CTCs from different cancer cell lines irrespective of their surface biomarker expression. Besides capturing CTCs, this system could also simultaneously perform the molecular profiling of the isolated CTCs. Since the phenotype of CTCs is highly heterogeneous, molecular profiling of CTCs at single-cell resolution is crucial for cancer diagnosis, therapeutic assessment, and studying metastasis progress. However, existing single-cell protein assay methodologies (e.g., flow cytometry, droplet microfluidics, microwell array) have significant drawbacks that hinder the multiplex biomarker analysis of individual CTCs, such as being time-consuming, requiring expensive instrumentation and trained personnel, being unable to process the sparse numbers of CTCs in a blood sample, and requiring sample enrichment protocols that could lead to loss of CTCs. In this novel microfluidic system, the simultaneous analysis of several biomarkers in a simplified procedure was made possible by the dual capability of magnetophoresis and the optical identification. When integrated with a single-cell fluorescence detection (SCFD) device, this platform efficiently collected composite spectral signatures of single CTCs free from any fluorescence overlap. This guaranteed accurate biomarker identification and permitted quantitative expression level profiling, resulting in a thorough phenotypic profile of individual CTCs. Figure 6(D) demonstrates this platform’s ability to perform multiplex biomarker analysis of CTCs at a single cell level in conjunction with the isolation of CTCs. Thus, this device overcame the drawbacks of current barcoding methods and provided a reliable solution for the in-depth study of CTCs. This advancement lowers the cost of multiplex phenotypic profiling while also simplifies the operational complexity. As a result, it can significantly increase the precision and dependability of CTC analysis in research and clinical contexts, opening the door to better oncology diagnostic and treatment approaches.

Figure 6.

Multiplex phenotypic profiling of CTCs separated with OEMBNs. (A) Capture efficiencies of MCF-7 cells from artificial blood samples (n = 3). (B) Fluorescent images of CTCs identified via immunocytochemistry. (C) Quantification of CTCs in patient blood samples. (D) 3D scatter plot of the fluorescence intensities of EGFR, HER2, and EpCAM receptors on individual CTCs. Reproduced with permission,(Wu et al., 2020) under a Creative Commons license CC BY 4.0.

Additionally, most CTC separation approaches conventionally utilize antibodies to target the surface receptors on CTCs. However, a limited number of antibodies can be conjugated to the IMNs due to their large molecular size, and the paratopes of the antibodies are usually randomly oriented, which depletes the antigen binding capacity of the IMNs. In this regard, several researchers have investigated various biomarkers that are smaller than antibodies and have a similar or better binding affinity to the surface receptors on CTCs. For example, Li et al. developed magnetic halloysite nanotubes (MHNTs) coated with β-cyclodextrin (β-CD) and a PEG-linker conjugated with FA that targets the FA receptor, a glycoprotein overexpressed on cancer cells.(Li et al., 2019) FA was utilized as a capture probe in this study, as it is affordable, non-immunogenic, non-toxic, highly stable, and has few functional groups for better orientation on the IMNs. MHNTs, composed of trivalent and divalent iron salts, contained surface amino groups that were covalently bonded to carboxyl-β-CD and then, they were coated with a PEG linker to attach FA onto the surface of the nanotubes. The PEG linker enhanced the stability of IMNs, reduced non-specific adsorption, and increased the availability of FA to capture Skov3, Hela, and A549 cancer cells. After incubating the three cancer cell lines with the prepared FA-functionalized nanotubes for 30 to 40 minutes, 95% of CTCs were captured from whole blood at a concentration of 100 cells/mL. Also, MHNTs could overcome a few flaws faced by the traditional Fe₃O₄ IMNs, including aggregation and leaky magnetism. Moreover, this approach could be adapted for various types of CTCs when the tumor surface markers are identified.

Another type of targeting ligands that are gaining attention by the scientific community are aptamers. Aptamers are single-stranded RNA or DNA molecules that have a high affinity for a set target antigen.(Zamay et al., 2020) They have several advantages over antibodies including high stability, high specificity, high flexibility, low production cost, no batch-to-batch variation, ability to bind to both small and large targets, and minimal immunogenicity. Aptamers also improve the biocompatibility and biological effectiveness and reduce the cytotoxicity of IMNs.(Jo & Ban, 2016; Mattarozzi et al., 2022; Zamay et al., 2020) Ding et al. developed a nanoparticle platform for the efficient isolation and ultrasensitive detection of CTCs using magnetic nanoparticles coated with leukocyte membranes combined with MUC-1 aptamer functionalized Ag₂S nanodots, as shown in Figure 7.(C. Ding et al., 2020) Initially, the hybrid membrane (HM) was created by fusing WBC and tumor cell membranes modified with SA, which was deposited on the surface of SiO₂-coated Fe₃O₄ IMNs. Then, multivalent, biotinylated aptamer-Ag₂S (Tetra-DNA modified) nanodots were attached to the HM-coated IMNs. The nanocomposites targeted the MUC-1 protein, which is overexpressed on the surface of MCF-7 breast cancer cells. The capture efficiencies of this nanocomposite in spiked lysed blood and whole blood samples were 96.24% and 90.25%, respectively. These nanoparticles captured CTCs from blood samples of cancer patients with a CTC concentration ranging from 6–10 cells per mL and did not capture any CTCs in blood samples of healthy donors.

Figure 7.

Schematic illustrating the preparation of HM-Fe₃O₄@SiO₂/Tetra-DNA-Ag₂S IMNs and their application in the efficient isolation of CTCs. Reproduced with permission,(C. Ding et al., 2020) under a Creative Commons license CC BY 4.0.

In another study, Li et al. developed anionic, 155 nm-sized IMNs functionalized with dual aptamers targeting EpCAM and protein tyrosine kinase 7 (PTK7) receptors that were highly capable of differentiating between CTCs that expressed either the EpCAM or the PTK7 receptor.(Li et al., 2022) At a concentration of 0.5 mg/mL, these IMNs could capture more than 95% of MGC-803 (high EpCAM expression) and BGC-823 (high PTK7 expression) gastric cancer cells and only 4% of THP-1 monocyte cells within 20 minutes of incubation, as shown in Figures 8(A) and (B). The IMNs could capture as low as 5 CTCs from mixed cell suspensions in PBS and lysed blood media, as shown in Figures 8(C) and (D). Immunocytochemistry could also distinguish the captured CTCs from the THP-1 cells in mimic whole blood, as shown in Figure 8(E). Dual-aptamer-modified IMNs also exhibited significantly higher capture efficiencies than single EpCAM- or PTK7-modified IMNs in mixed-cell suspensions and blood samples from three gastric cancer patients. These authors also conducted gene analysis of the isolated CTCs, where they determined that a high expression of PTK7 was indicative of a poor survival prognosis and there was a significant negative correlation between EpCAM and PTK7 expression, indicating that PTK7 was a reliable surface biomarker for mesenchymal CTCs. In addition, MGC-803 cells had a higher expression of multidrug resistance-associated proteins (MRP1, MRP3, MRP4, and MDR1) than that of BGC-823 cells, indicating that MGC-803 cells were not as susceptible to chemotherapeutic drugs as BGC-823 cells.

Figure 8.

Separation of MGC-803 and BGC-823 cells using dual-aptamer modified IMNs. (A) Optimization of IMN concentration and (B) incubation time. Capture efficiencies of (C) MGC-803 and (D) BGC-823 cells in different media. (E) Identification of isolated CTCs by immunocytochemistry. Reproduced with permission,(Li et al., 2022) under a Creative Commons license CC BY 4.0.

Given the phenotypic heterogeneity of cancerous tumors, there is no specific biomarker that can be used to target different subpopulations of CTCs. Thus, researchers have focused on developing separation methods independent of surface receptor expression on CTCs. For instance, Chang et al. fabricated biomimetic IMNs cloaked with a hybrid cancer cell membrane-liposome membrane (CM-LMs) to recognize and capture CTCs in whole blood samples.(Chang et al., 2020) Since cell adhesion molecules are overexpressed on cancer cell membranes, CTCs can self-target and adhere to their homologous cells. Thus, by combining a liposome membrane with a cancer cell membrane, CM-LM-IMNs identified specific homologous CTCs and demonstrated a markedly decreased affinity for normal cells or other heterogeneous CTCs. As a result, CM-LM-IMNs outperformed traditional IMNs coated with antibodies in terms of CTC capture efficiency (75% within 30 minutes). This study also demonstrated that CM-LM-IMNs obtained a greater detection rate and purity (8.8% leukocytes captured) of CTCs from orthotopic 4T1 mice metastatic mammary cancer models compared to the conventional anti-EpCAM-based immuno-IMNs. Moreover, the cell membrane coatings’ biomimetic qualities enabled more effective and precise targeting of CTCs, as they were responsible for the improved performance of these materials. Another reason for utilizing a cancer-cell membrane hybridized with a liposome membrane is that the captured CTCs can be released by adding a trypsin- ethylenediaminetetraacetic acid (EDTA) solution. This overcomes one of the main disadvantages that conventional IMNs face: damage to cells caused by chemicals or mechanical forces used to separate captured CTCs from IMNs for further clinical analysis. More specifically, these authors could recover over 85% of CTCs released from CM-LM-IMNs within 2 minutes after they were treated with trypsin-EDTA.

Finally, in a different study, Liu et al. developed a novel technology called integrated ferrohydrodynamic cell separation (iFCS) to isolate CTCs independent of their surface antigen expression or physical characteristics in blood samples with a throughput of 6 mL per hour.(Yang Liu et al., 2021) This method combined the principles of magnetophoresis and diamagnetophoresis of blood cells to simultaneously deplete WBCs and isolate CTCs, respectively. The iFCS device was filled with ferrofluids, allowing both magnetophoresis and diamagnetophoresis to coexist simultaneously, providing tunable control over the environment. This device depleted over 99% of WBCs and recovered 94.99–99.68% of PC-3, MCF7, MDA-MB-231, HCC1806, H1299, H3122, Jones, DMS79, and H69 cells spiked in 1 mL of blood at a CTC concentration of 100 cells per mL, as presented in Table 2. Similarly, Zhao et al. also showcased an iFCS-based cell separation, with a high throughput of 12 mL per hour, a recovery rate of 99.08% in samples spiked with CTCs at concentrations ranging from 1–10 cells per mL, and a low WBC contamination of 533 cells per mL of blood processed.(Zhao et al., 2019) These two studies demonstrate the potential of the iFCS device as a minimally invasive alternative for studying metastatic cancers. This device utilizes biocompatible ferrofluids for a comprehensive enrichment and analysis of viable CTCs, potentially improving the development of diagnostic and therapeutic strategies.

4. Magnetic isolation and analysis of fetal and abnormal RBCs for prenatal testing and anemia screening

In the previous sections, we primarily elaborated on how antigen-specific magnetic nanoparticles can separate target cells from a blood sample using a labeled-based procedure. This section will detail how magnetism can be utilized to separate fetal and abnormal RBCs from normal, healthy RBCs for diagnostic purposes using both labeled and label-free techniques. Indeed, for some conditions, unlabeled separation via magnetophoresis can be used to isolate weakly paramagnetic cells instead of using magnetic nanoparticles to target specific surface antigens on RBCs or diamagnetophoresis via ferrohydrodynamic isolation devices for unlabeled separations. Physiological events such as differentiation, maturation, cell infection, cell aging, or death can modify the cells’ physical traits.(Durmus et al., 2015a) One such trait is the magnetic signature of cells; RBCs display a unique paramagnetic behavior when the Hb is deoxygenated or oxidized, which varies as a function of their amount/disease state, and this enables them to be separated from other cells under a magnetic field gradient.(Nemescu et al., 2020) Another such trait is the density of the cells; abnormal RBCs in certain types of anemia have a higher density than normal RBCs, thus the cells can be separated using magnetic levitation.(Knowlton et al., 2015; Yenilmez et al., 2016) Table 3 presents the recent developments in isolating fetal and abnormal (anemic, sickle, etc.) RBCs via magnetophoresis exploiting the differences in these physical characteristics as well as using conventional labeled approaches.

Table 3.

Magnetophoresis of fetal and abnormal RBCs for disease detection and prenatal screening.

Disease	IMNs characteristics (for labeled separations)	Sample	Magnetic separator	Continuous or batch	Volume/flow rate	Quantitative Separation factors	Other comments	Ref
Sickle cell	Label-free	Blood samples from sickle cell patients and healthy donors	Two N52-grade neodymium bar magnets	Batch	30 μL	Accuracy: >85.7%	Magnetic levitation - label-free system. RBCs equilibrated in non-toxic and non-ionic paramagnetic medium (gadolinium)	(Goreke et al., 2022)
Malaria	Label-free	RBCs from whole blood samples of healthy patients, treated with sodium nitrite to model infected RBCs	Two types of N42 and N52 NdFeB magnets (590–610 kA/m and 6–7×1014 A2/m3)	Continuous	8 mL/min	Capture efficiency: 52–70%	Ni posts were etched on a silicon wafer with gold electrodes for impedance measurements. After 10 minutes of processing, nonspecific adsorption of untreated RBCs took place. Capture efficiencies were quantified with fluorescence microscopy	(Marco Giacometti et al., 2022)
Malaria	Label-free	Whole blood from infected patients and healthy donors	Two N52-grade neodymium bar magnets (600 kA/m and 7×1014 A2/m3)	Batch	70 μL	Specificity: 49.2–84.7%; Sensitivity: 93.3–100%	Ni cylindrical micro-concentrators etched on a silicon wafer with gold electrodes for impedance measurements. Detection sensitivity of 10 RBCs per μL and a dynamic range up to 105 RBCs per μL	(Giacometti et al., 2021)
Malaria	Label-free	RBCs from whole blood samples of healthy patients, treated with sodium nitrite to model infected RBCs	Two N52-grade neodymium bar magnets (7*1014 A2/m3)	Batch	-	-	Ni concentrators embedded in a silicon substrate with gold electrodes for impedance measurements. High magnetic field gradient in a lab-on-chip platform that could detect both hemozoin crystals and infected RBCs	(Milesi et al., 2020)
Fetal nucleated RBCs	Fe3O4 IMNs coated with a hybrid RBC/WBC membrane and functionalized with anti-CD147 antibody (Core diameter: 200 nm; Shell thickness: 10 nm)	Clinical maternal blood (11–13 gestational weeks)	-	Batch	2 mL	Capture efficiency: >90%; Purity: 87%	Captured 18 fetal RBCs from 1 mL of maternal blood. The concentration of the isolated cells ranged from 11 to 24 cells in 1 mL of blood. Fluorescence in situ hybridization (FISH) analysis was carried out to detect fetal chromosome aneuploidies (21/Y, 13/X, 18)	(Zixiang Wang et al., 2021)
Fetal nucleated RBCs	Label-free and labeled separation (CD71 targeting IMNs)	Maternal blood (mean gestational age of 21 weeks)	VarioMACS™ and MiniMACS MS+ columns (Miltenyi Biotec) (1.4 T)	Continuous for label-free separation and batch for labeled separation	270 mL/s for the label-free and 1.5 mL for the labeled separation	Capture efficiency: 96% for label-free, 98.6% for labeled isolation	FISH analysis detected at least one XY RBC in 81.5% and 61.5% of samples for the label-free and the labeled isolation, respectively	(Nemescu et al., 2020)
Malaria (cells at different stages)	Label-free	Healthy RBCs treated with sodium nitrite	Two neodymium permanent magnets (300 mT)	Batch	30 μL	Up to 2000-fold enrichment in a high-density medium	Magnetic levitation distinguished both late-stage RBCs and younger ring-stage cells. Ring-form RBCs levitated higher than uninfected RBCs	(Deshmukh et al., 2021)
Malaria	Label-free	Whole blood from malaria-positive patients. RBCs lysed with 0.1% saponin and suspended in 1 mM gadobutrol	Two neodymium permanent magnets (300 mT)	Batch	50 μL	Accuracy: 95%; Sensitivity: 91%; Specificity: 100%	Portable smartphone-based magnetic levitation platform for sample imaging and parasite detection	(Deshmukh et al., 2022)
Malaria	Label-free	Mouse blood samples	Two rows of permanent magnets (0.4 T)	Continuous	Variable flow rates (0.4–0.8 mL/min)	Capture efficiency: 9.1–25%	Microfluidic chip with nickel microstructures (square, V-shaped, or W-shaped) placed on a copper sheet. Low flow rates were first used to capture the cells and then increased to wash away the beads and normal cells	(Noosawat et al., 2022)
Mature gametocytes from asexual ring-stage parasites	Label-free	NF54 gametocyte material spiked in peripheral blood	QuadroMACS separator and LS column (Miltenyi Biotec)	Continuous	0.67 mL/min	Capture efficiency: 96.33–99.11% for gametocytes, 0.64% for ring-stage parasites	Less than 5% of gametocytes appeared in the eluted cells. The binding efficiency of reused columns was also investigated, and the column retained its binding efficiency for 7 separate cycles	(Graumans et al., 2019)
Sickle cell disease	Label-free	RBC samples from healthy donors and patient’s apheresis waste	Quadrupole magnetic sorter (NdFeB permanent magnets, 1.36 T and 286 T/m)	Continuous	0.5 mL/min	-	Fractionated RBCs based on their Hb content. Enriched outlet had a greater p50 value than the feed and non-enriched outlet, indicating the presence of sickle RBCs that cannot bind to O2	(Mitchell Weigand et al., 2022)
Sickle cell disease	Label-free	Whole blood from healthy donors and patients (transfused and non-transfused) and apheresis product	NdFeB permanent magnets (212.77 T/mm2)	Batch	2 mL	-	Cell tracking velocimetry measured the magnetically induced velocity and gravity-induced settling velocity for oxygenated, deoxygenated, and oxidized RBCs. Used to measure cell size, Hb content, and their distributions to test its ability to diagnose sickle cell disease	(Gómez-Pastora, Weigand, et al., 2022)
Sickle cell disease	Label-free	Whole blood from healthy donors and patients (transfused and non-transfused) and apheresis product	NdFeB permanent magnets (212.77 T/mm2)	Batch	2 mL	-	Cell tracking velocimetry was employed to analyze oxygenated, deoxygenated, and oxidized RBCs. Measured oxygen equilibrium curves under intermediate O2 levels and the ability of the instrument to differentiate sickle from healthy RBCs	(M. Weigand et al., 2022)
Anemia and iron deficiency	Label-free	Healthy whole blood samples	NdFeB permanent magnets (212.77 T/mm2)	Batch	2 mL	-	Used cell tracking velocimetry to determine the single-cell Hb content of RBCs (29.2 pg Hb per cell in healthy samples) to determine the device’s diagnostic power for anemia and iron deficiency	(Kim et al., 2020)
Malaria (infected RBCs)	Label-free	Bovine RBCs treated with sodium nitrite	Two N42 grade NdFeB permanent magnets (>1014 A2/m3)	Batch	70 μL	-	Lab-on-a-chip device with Ni concentrators embedded in a silicon substrate with gold electrodes for impedance measurements. The detection limit was 40 parasites/μL in 10 minutes	(M. Giacometti et al., 2022)
Fetal blood cells	Carboxyl-Fe2O3 polystyrene IMNs and quantum dots functionalized with anti-CD71 and anti-γ-globin (size: 150–200 nm)	Maternal blood samples	-	Batch	10 mL	Fetal cells detected in 24/25 samples (on average, 10.1 cells detected)	Anti-CD71 conjugated IMNs for separation and anti-γ-globin conjugated quantum dots for identification. Fetal cells not detected in non-maternal samples	(Sui et al., 2019)

Fetal nucleated RBCs are of value to clinicians, as they can give an insight into the fetal genome, which is invaluable for prenatal screening. Due to their rarity and variability, it is important that the methods used to isolate fetal RBCs from blood are simple, efficient, and reproducible. Maternal blood samples are usually pre-processed using a double-density gradient centrifugation method followed by a washing step to collect the cells at the gradient interface. However, magnetic tools have also been explored for fetal cell isolation. For instance, Nemescu et al. compared the isolation efficiencies of two magnetic enrichment techniques; one was a stationary capture of RBCs in a magnetic column based on the paramagnetic properties of methemoglobin (NaNO₂ was used to induce paramagnetism in the nucleated cells), and the other was a labeled separation approach using magnetic beads functionalized with anti-CD71.(Nemescu et al., 2020) In this study, the number of isolated fetal RBCs were 29.7×10⁴ and 10.1×10⁴ with the stationary magnetic column and the anti-CD71 functionalized magnetic beads, respectively. After processing, viable cells from both methods were counted, fixed, and prepared on slides via cytocentrifugation. Fluorescence in situ hybridization (FISH) was then carried out to identify fetal RBCs by determining the Y chromosome. The slides were stained to assess the Hb-rich cells, but treatments with benzidine and May-Grunwald/Giemsa were omitted to preserve the sample’s quality for FISH. At least one XY cell was found in 81.5% and 61.5% of blood samples treated with the magnetic column and with anti-CD71 functionalized magnetic beads, respectively. The study’s findings highlighted the differences in cell yield and efficiency between Hb-based paramagnetic selection and anti-CD71 antibody sorting, suggesting significant implications for prenatal diagnostic applications.

Sickle and anemic RBCs are other RBCs that can be fractionated by magnetophoresis for a variety of applications, including diagnosis, disease management, and to avoid adverse effects of blood transfusions performed with low-quality and low-Hb RBCs. For example, some patients with sickle cell disease require regular RBC exchanges, a type of blood transfusion where all the patient’s RBCs are removed from the patient’s circulation using an apheresis machine and then replaced with non-sickle RBCs from healthy donors. In this regard, magnetophoresis can be used to remove only the sickle RBCs from the patient’s blood while returning to the patient’s circulation non-sickle RBCs, which are still functional, avoiding the detrimental consequences of repeated RBC transfusions while at the same time reducing the amount of RBC units needed for the process, increasing the RBC unit supply for other therapies. Weigand et al. explored the continuous-flow magnetic fractionation of healthy, aged, and/or sickle RBCs using a pressure-driven sorting system, investigating how factors such as flow rates, buffer compositions, and the inherent characteristics of RBCs influence the magnetophoresis.(Mitchell Weigand et al., 2022) RBC samples were collected from healthy donors and sickle cell disease patients undergoing RBC exchange transfusions. These samples were processed through a flow system using PEG-enhanced buffers to improve separation efficiency. These authors also conducted computational simulations of RBC trajectories under different magnetic and fluidic conditions within the separator. The analysis of the feed and sorted fractions involved measuring size distributions, magnetic susceptibilities, Hb content of single cells, and assessing oxygen saturation levels, among others. Their research demonstrated the potential of continuous-flow magnetic fractionation systems to process large volumes of RBCs while addressing crucial issues like lift forces and buffer homogeneity. The findings revealed a major challenge in achieving complete magnetophoresis due to the overlapping characteristics between enriched and non-enriched fractions. This study highlights the importance of optimizing buffer oxygenation to enhance the specificity of magnetic sorting, particularly when handling heterogeneous samples such as sickle cell disease blood. It also addressed the trade-offs between throughput and purity, underscoring the necessity for further refinements in buffer composition and system parameters to achieve a more effective magnetic fractionation for clinical applications.

Additionally, RBCs infected with malaria also exhibit paramagnetism; when the Plasmodium parasite feeds on Hb, heme is released and converted to insoluble hemozoin crystals, a biomarker widely used for detecting malaria. Many studies have been carried out to separate infected RBCs (i-RBCs) from healthy RBCs (h-RBCs) by exploiting the differences in their magnetic susceptibilities. As shown in Figures 9(A) and (B), Giacometti et al. developed a novel approach for malaria detection in blood samples, a lab-on-a-chip method (TMek), that employed micromagnetic nickel concentrators on a silicon microchip for the precise capture of i-RBCs and the quantification of the level of parasites.(Giacometti et al., 2021) External magnets generated a high magnetic field gradient that attracted and selectively captured i-RBCs in the microfluidic chamber, and at the same time, differential impedimetric detection measured changes in electrical impedance caused by captured i-RBCs at various stages of parasite development. The impedance signal was proportional to corpuscle concentration, facilitating accurate parasitemia quantification and stage identification without active microfluidic manipulation. Validation synthetic models, with known concentrations of treated RBCs (t-RBCs) mimicking i-RBCs, demonstrated a linear relationship between signal amplitude and t-RBC concentration and improved sensitivities at longer capture times. The lowest detectable concentrations for capture times of 5 and 10 minutes were 100 t-RBCs per μL and 10 t-RBCs per μL, respectively. Preclinical studies performed with samples from 75 malaria patients demonstrated that this device had 100% specificity and 69% sensitivity with respect to microscopy and clinical data. Experiments with cultivated Plasmodium falciparum i-RBCs further validated the device’s capability to distinguish among the three parasite stages (ring, late trophozoite, and gametocyte) based on their distinct physical properties and magnetic susceptibilities.

Figure 9.

(A) Conceptual representation of the magnetophoretic separation of malaria i-RBCs: infected cells are captured by nickel microposts on a microchip when subjected to an external magnetic field, while the h-RBCs settle on a glass slide. The scheme also presents the main forces acting on infected and healthy cells within the device. (B) Microchip with the measurement (red) and reference (blue) areas on the left and top view of the annular gold electrodes on the Ni magnetic concentrators on the right. Reproduced with permission,(Giacometti et al., 2021) under a Creative Commons license CC BY 4.0.

In another study, Giacometti et al. developed and optimized another lab-on-a-chip device to capture magnetic, malaria i-RBCs.(Marco Giacometti et al., 2022) In their system, i-RBCs were captured by nickel posts, and h-RBCs settled on a glass slide due to gravitational forces. This investigation validated the effectiveness of nickel posts in generating robust magnetic field gradients necessary for capturing RBCs exhibiting a high magnetic susceptibility (in this study due to methemoglobin formation after treating healthy RBCs with sodium nitrite, thus mimicking i-RBCs). Experimental protocols included precise manipulation of various sample volumes and magnet configurations to optimize conditions for magnetophoretic capture experiments. Utilizing computational simulations, this study optimized the geometry of Ni posts and the strength of external magnetic fields. Their simulation findings were further substantiated through experimental validations using bovine blood models, confirming the chip’s separation efficacy of RBCs replicating malaria-infected ones. It was determined that capture efficiencies of 70% and 52% were achieved in 10 minutes from blood samples drawn after three hours and seven hours, respectively, in a chip with 20-μm-tall nickel posts.

Additionally, magnetic levitation is a novel magnetic-based manipulation technology that has garnered significant interest in recent years, where magnetic fields can manipulate the spatial position of diamagnetic cells in a capillary microchannel based on the differences in their density profiles and magnetic properties.(Karakuzu et al., 2024) In one study, Deshmukh et al. developed a levitation device to separate malaria-i-RBCs from healthy RBCs that comprised two neodymium permanent bar magnets with dimensions of 5 cm × 0.5 cm × 0.2 cm and surface magnetic field strengths of 300 mT, arranged in an anti-Helmholtz configuration with like poles facing each other.(Deshmukh et al., 2021) These magnets were securely held in place using polymethyl methacrylate plastic pieces, which also accommodated angled mirrors for vertical light path imaging. A square glass capillary (1 mm × 1 mm × 5 cm, with 0.2 mm thick walls) was used to hold the cell suspension mixture, as shown in Figure 10(A). This capillary was positioned within the 1 mm gap between the magnets, enabling controlled cell levitation, and the entire setup was mounted on a standard microscope stage for imaging. The platform’s design ensured consistent levitation of RBCs at heights, which enabled precise density-based analysis at the single-cell level. As shown in Figure 10(B), h-RBCs and i-RBCs were suspended in paramagnetic solutions containing chelated gadolinium ions to achieve optimal magnetic susceptibility differences between the cells and the medium for the levitation experiments. After 15 minutes, the final vertical positions of the cells were imaged and analyzed to construct a height distribution curve or levitation pattern of ring-stage i-RBCs and h-RBCs, as shown in Figure 10(C). It was determined that the levitation profile of the i-RBCs was distinct from the h-RBCs in terms of the shape of its distribution and the mean equilibrium height. The integration of computational modeling, experimental validation, and image analysis techniques in their study paves the way for further advancements in the characterization of the cells’ biophysical signatures and diagnosis using rapid, label-free, and non-invasive methodologies.

Figure 10.

Identification of i-RBCs in blood samples using magnetic levitation. (A) A small volume of blood, less than a fingerprick, is mixed with a paramagnetic solution inside a glass capillary and loaded into the magnetic device. Scale bar = 1 cm. (B) The cells start to levitate at various heights, reaching equilibrium within 12–16 minutes, resulting in a height-based separation of different cell types. (C) The device’s schematic shows h-RBCs levitating in the upper half of the channel due to buoyancy and gravity since the cells are slightly less dense than the medium. Since h-RBCs are diamagnetic, they move away from the high magnetic gradient near the top magnet due to a repulsive magnetic force. i-RBCs are RBCs with ring-forms of the parasite, which are believed to decrease the density and increase the magnetic susceptibility of the host RBCs. This increases buoyancy and reduces the effects of gravity and magnetic repulsion, causing parasite-containing RBCs to levitate higher (z-axis) than the uninfected RBCs. In the figure, h represents levitation height (μm), χ denotes magnetic susceptibility (unitless), and ρ indicates density (g/mL). Reproduced with permission,(Deshmukh et al., 2021) under a Creative Commons license CC BY 4.0.

Building on this foundation, Deshmukh et al. developed a portable and automated platform to detect malaria parasites utilizing magnetic levitation combined with automated imaging and artificial intelligence. (Deshmukh et al., 2022) The images were then analyzed using convolutional neural network for the accurate detection of the malaria parasites. This automated system reduced the need for expert manual microscopy, speeding up diagnosis and minimizing human error. The detection system’s performance was highly accurate: it correctly identified all malaria-positive cases (100% sensitivity) and correctly recognized 91% of malaria-negative cases (specificity), meaning there were very few false negatives or false positives compared to gold-standard diagnostic methods like microscopy and PCR. Additionally, it could detect very low levels of infection, down to 31 parasites per microliter of blood, which is important for early diagnosis and controlling disease spread.

In a similar study, Goreke et al. introduced the MagDense platform for fractionating sickle from healthy RBCs by exploiting the differences in their densities, a significant advancement in disease screening.(Goreke et al., 2022) As shown in Figure 11(A), this platform comprised of a levitation channel with a 1 mm × 1 mm cross-sectional area situated between two powerful Nd52-grade magnets with a magnetic field strength of 1.4 T. RBCs suspended in a paramagnetic medium levitated within this channel until the magnetic and buoyancy forces were in equilibrium with each other, as shown in Figure 11(B). Validation using polyethylene beads of known densities demonstrated the platform’s high accuracy, achieving a linear relationship between the density and levitation height, showcasing its capability for precise and reliable density measurements. The MagDense platform was further validated in clinical contexts, particularly in fractionating sickle (HbS) RBCs from healthy (HbA) cells, taking advantage of the fact that sickle Hb (HbS)-containing cells have a higher average cell density than normal Hb (HbA)-containing cells. Hundreds of individual RBCs were analyzed, revealing heterogeneous density distributions among HbS-RBCs indicative of the disease’s cellular variability. The platform’s ability to differentiate between HbS- and HbA-containing RBCs was highlighted by a receiver operating characteristic (ROC) curve analysis, achieving a minimum accuracy of 85.7%. Moreover, the authors introduced the “RBC levitational density width (RLDW)” as a new biophysical marker, which further enhanced the system’s utility in quantifying density distribution patterns, akin to established parameters like the RBC distribution width (RDW) in blood counts. These findings emphasize the MagDense platform’s potential as a valuable tool for precise disease monitoring through detailed RBC density characterization.

Figure 11.

(A) The MagDense platform consists of a microchannel situated between two permanent magnets. Inclined mirrors offer real-time imaging and facilitate efficient evaluation of blood samples. (B) A diluted mixture of sickle and healthy RBCs is loaded into the microfluidic device and left to equilibrate under the influence of gravitational and magnetic levitation forces within a paramagnetic medium. Reproduced with permission,(Goreke et al., 2022) under a Creative Commons license CC BY 4.0.

5. Current challenges and future prospects in the field

Detecting rare cells in blood samples plays a significant role in providing insights into the mechanisms of specific diseases or for developing personalized treatments for patients. Traditional methods for separating cells, such as Ficoll-Paque density gradient centrifugation, fluorescence activated cell sorting, or size-based filtration approaches, face significant challenges in detecting rare cells. These challenges include limitations in achieving a high-resolution separation, the large blood volume required to be processed in order to detect only a few of these rare cells, the high cost of these technologies, the need for trained personnel to operate them, a high contamination from other cells in the isolated fraction, and potential cell damage.(Yu et al., 2014)

In recent years, research has focused on using magnetophoresis to isolate rare cells from blood. Magnetophoresis is the underlying process in magnetic separation, where the motion of biomolecules is manipulated with an external magnet with a high magnetic field strength and field gradient. The advantages over conventional techniques include simplicity, fast processing time, high separation efficiency, cost-effectiveness, high robustness, and low energy consumption.(Leong et al., 2016; Wu et al., 2023; Xian Wu et al., 2022) In magnetophoretic-based systems, two main approaches are commonly applied: labeled and label-free magnetic separation. Table 4 provides a comparative overview of both labeled and label-free magnetic separation approaches highlighting representative examples, performance metrics, clinically available technologies, and key technological challenges associated with each method.

Table 4.

Comparison of label-free and labeled magnetic separation technologies - examples, performance metrics, commercial technologies, and key challenges.

Approaches	Examples	Targeting ligands	Target Cell Types	Performance Metrics	Commercial clinical technologies (Descamps et al., 2022)	Technological Challenges (Aubry et al., 2023; Frenea-Robin & Marchalot, 2022a; Rushton et al., 2021; Wongkaew et al., 2019; Zhang et al., 2011)
Labeled	Nanoparticles coated with polymers/cell membranes	EpCAM antibody or aptamer	CTCs	Capture efficiency: 40–98%; Purity: 34–96.3%; Viability: 80–99.3%	IsoFlux™ (Fluxion Biosciences) LiquidBiopsy® (Cynvenio Biosystems) CellSearch® (Menarini-Silicon Biosystems) MagSweeper (Stanford University) EasySep™ EpCAM Positive Selection Kit (STEMCELL Technologies)	Low to moderate specificity: downregulation of EpCAM expression after epithelial-to-mesenchymal transition Low to moderate sensitivity: variability in EpCAM expression across different cancer types and patient samples or very rare expression of RBC markers Low to moderate capture purity and yield: unstable ligand immobilization and nonspecific binding to other blood cells Limited testing of the IMNs’ biocompatibility: uncertainty over safe in vivo applications Limited release methods that preserve the cells’ viability and functions for downstream analysis Limited quality, reproducibility, and scalability for clinical use
		Non-EpCAM based ligands (antibodies, aptamers, small molecules, peptides)	CTCs	Capture efficiency: 40.85–100.5%; Purity: 36–99.6%	AdnaTest (QIAGEN) Dynabeads™ Epithelial Enrich (ThermoFisher Scientific)
		Anti-CD147, anti-CD71, anti-γ-globin antibodies	RBCs	Capture efficiency: 90–98.6%	-
Label-free	Nanoparticles-coated with polymers (Charge based separation)	-	CTCs	Capture efficiency: 66–97%; Purity: 66–91.2%	-	Achieving reproducible, stable, uniform coatings: variable separation efficiency Nonspecific adsorption of proteins and other biomolecules: variable capture specificity Batch to batch variability and difficulty in maintaining surface chemistry stability and colloidal stability: impediments to large scale manufacturing
	Lab-on-a-chip device with Ni concentrators embedded in a silicon substrate with gold electrodes	-	RBCs	Capture efficiency: 17.05–66.95%	-	Complex microfabrication techniques Difficult to achieve consistent and precise geometry of the Ni concentrators Limited sensitivity and specificity due to small electrode size, background noise, and varying cell properties
	Magnetic column separators		RBCs	Capture efficiency: 96–97.72%	MACS® Columns (Miltenyi Biotec)	Reduced purity of RBCs due to contamination from other blood components Generating a consistent and strong magnetic field within the column to ensure uniform RBC capture and minimize loss Inability of high throughput blood processing due to clogging and fouling at high cell densities Achieving reproducible separation results across different devices, blood samples, and after multiple uses
	Integrated ferrohydrodynamic chip	-	CTCs and RBCs	Capture efficiency: 97.34–99.08%	-	Advanced fabrication techniques Complex precise control over magnetic force field uniformity and strength Optimize flow control to maintain cell viability and effective separation Maintaining consistent fabrication is difficult affecting device reproducibility Ensuring biocompatibility of ferrofluids and magnetic materials to preserve RBC functions
	Magnetic Levitation	-	CTCs and RBCs	Capture efficiency: 85.7–95% and 2,000-fold enrichment	-	Data processing requires advanced algorithms Difficult to ensure consistent cell conditions Precise material and device design for creating stable magnetic fields Requires very sensitive system calibration to differentiate between normal and abnormal cells Overlapping levitation bands make it difficult to identify distinct cell populations Unable to process large volumes of blood quickly

Labeled techniques involve the selective separation of target cells with magnetic nanoparticles functionalized with biological ligands under the application of a magnetic field. Many studies have been conducted on utilizing IMNs to isolate rare blood cells. IMNs are mainly composed of metals, such as ferrites, metal alloys, or iron-based magnetic oxides. Apart from their facile synthesis, functionalization, and characterization, IMNs 0077ith a size range of a few to tens of nanometers have superparamagnetic behavior, are stable in biological fluids, and have high specific surface areas that can encapsulate drugs or get conjugated with biological moieties, making them popular for diagnostics.(Qin et al., 2013) However, there still exist some limitations to using IMNs for biomedical applications.

A major challenge in the use of IMNs functionalized with biological ligands that bind to the target cells is the variability in their specificity and sensitivity. Most current studies and clinically available technologies rely on capturing CTCs via the EpCAM marker. However, the specificity of these approaches is compromised by the downregulation of EpCAM expression during epithelial-to-mesenchymal transition, which limits the isolation of clinically relevant, metastatic CTCs. Additionally, the heterogeneity in EpCAM and other biomarker expression across different cancer types and patient samples results in inconsistent sensitivity, thereby reducing the reliability of these technologies in diverse clinical contexts. Recent research efforts are therefore focused on overcoming these limitations through combinatorial targeting strategies to account for biomarker variability, engineering more stable and covalently immobilized ligands to enhance capture efficiency and purity, and optimizing surface coatings to reduce nonspecific binding.

Research is also being conducted on improving synthesis methods, as they face several limitations regarding reproducibility, scalability, and difficulty regulating particle size distribution, yield, and purity.(Rezaei et al., 2024) Since variations between different IMN batches can impact their performance in diagnostics, future research should be conducted to develop standardized protocols for the synthesis and characterization of IMNs for easy comparison between different studies and to ensure a smooth transition from laboratory research to clinical applications.(Rezaei et al., 2024) Additionally, studies on the biological synthesis of IMNs using green manufacturing methods have been recently performed, and the techniques reported hold great potential to improve the toxicity of the materials as well as to shift nanomaterial manufacturing processes toward more environmentally friendly and sustainable models. In addition, the development of novel materials with enhanced magnetic properties—such as superparamagnetism and high saturation magnetization—is critical for improving the magnetic forces exerted on labeled cells. Importantly, these materials should maintain strong magnetic responsiveness even at nanoscale dimensions (typically <50 nm), thereby ensuring a high surface-area-to-volume ratio. This characteristic not only enhances separation efficiency but also enables the use of lower particle dosages, contributing to improved process economy and reduced cytotoxicity.

From the overview and the analysis of the studies presented above, we noticed that various works emphasize particle size as a key determinant of capture efficiency; however, the observed trend (as shown in Figure 12) suggests that there is little correlation between particle size and capture efficiency of the target cells. This indicates that other factors—such as surface chemistry, magnetic properties, and functionalization—may play a more significant role in determining capture efficiency. More studies on particle manufacturing methods are required to establish optimal process conditions, increase production rates and yields, as well as to ensure reproducibility and consistency of the physicochemical properties of the nanomaterials. Further investigations should also focus on systematically evaluating these additional influencing factors to gain deeper insights and facilitate comparisons between studies, promoting meta-analysis investigations.(González-Fernández et al., 2024; Rezaei et al., 2024)

Figure 12.

Scatter plot showing the relationship between particle diameter and capture efficiency. Data points are obtained from references (Chen et al., 2019; L. Ding et al., 2020; Ding et al., 2021; Doswald et al., 2022; Esmaeilsabzali et al., 2019; Feng et al., 2020; Ghafouri & Badieirostami, 2021; Gou et al., 2021; Gribko et al., 2021; Haghighi et al., 2019; Hazra et al., 2020; M. Hu et al., 2022; Jiang et al., 2022; Li et al., 2022; Yang Liu et al., 2021; Meng et al., 2019; Meng et al., 2020; Mohammadi et al., 2022; Nasiri et al., 2022; Nian et al., 2024; Pan et al., 2022; Park et al., 2021; Payer et al., 2020; Sand et al., 2020; Seyfoori et al., 2019; Tian et al., 2022; Unni et al., 2020; Wang et al., 2023; Zixiang Wang et al., 2021; Zhili Wang et al., 2021; Wavhale et al., 2021; Wu et al., 2020; Xu et al., 2023; F. Zhang et al., 2019; L. Zhang et al., 2019; P. Zhang et al., 2021; Y. Zhang et al., 2021; Zhang et al., 2023; Zhang et al., 2022; Zhou et al., 2019)

Another area for improvement is the need for studies investigating the toxicity of IMNs in vivo and in vitro. The accuracy as well as the results and findings of toxicity reports depend on several factors such as the particle’s chemical composition, size, shape, surface chemistry, dose administered, method of administration, pharmacokinetics, bioavailability, and more.(Tran & Webster, 2010) If the IMNs are toxic to the isolated cells, it will be challenging to carry out further clinical analysis on the cells. Thus, before they are applied for clinical use, both in vivo and in vitro cytotoxic studies must be carried out to fully comprehend the toxicity of IMNs and to ensure the safety of the nanomaterials.(Cardoso et al., 2018)

Moreover, IMNs can be combined with microfluidic devices and sensor technology to create miniaturized diagnostic systems that are rapid, portable, cost-effective, and accurate. Microfluidic devices offer numerous advantages over conventional, bulky diagnostic equipment, including lower sample volumes required, shorter analysis times, and reduced biological and chemical waste generation.(Kim et al., 2023) However, microfluidic devices still face significant challenges that need to be addressed in the near future, such as laborious fabrication, intricate design, and complex control and operation.(Yu et al., 2014) Future research areas within microfluidics should include integrating multiple separation modules to isolate multiple cell types based on various markers, and upstream cell sorting with downstream cellular multiplex analysis on a single-chip platform in a fully automated manner. Research can also be conducted on integrating advanced magnetic field configurations into microfluidic platforms to enhance the efficiency and specificity of magnetic capture under flow conditions. Additionally, comprehensive studies are needed to optimize key operational parameters—including flow rates and microchannel geometry—to improve both capture efficiency and the purity of isolated cells.

A common limitation of labeled magnetic separation technologies within diagnostic applications is that the rare cells are generally required to be bound to antibody-conjugated magnetic beads so that the cells can be magnetized and separated under a magnetic field gradient. After the process, these cells must be separated from the magnetic beads by physical forces or chemical methods for further downstream clinical analysis; however, these methods can damage the cells and their functions. Thus, research has focused on label-free magnetic-based separation technologies to overcome this challenge.

Magnetic column separators—such as the commercially available Miltenyi MACS^® columns—are an example of technologies categorized under high-gradient magnetic separation (HGMS) systems. These devices generate strong localized magnetic field gradients within columns packed with ferromagnetic materials, which serve to dehomogenize the externally applied magnetic field. (Strayer et al., 2025) As cells flow through the column, those labeled with magnetic particles experience enhanced magnetic forces near the ferromagnetic matrix and are subsequently retained. Key advantages of these systems include low cost, minimal energy requirements, rapid processing times, and biological non-invasiveness.

Despite these benefits, magnetic column separators are limited by challenges in separation purity, specificity, throughput, and cell viability, as outlined in Table 4. Purity and specificity are often compromised by nonspecific interactions between cells and the magnetic beads or the column matrix, as well as by similarities in the physical or biological properties of target and non-target cells. Additionally, reproducibility remains a concern, as results can vary between devices, sample types, and repeated uses. Future research should focus on optimizing the composition and surface coatings of magnetic beads to enhance binding affinity and selectivity, refining flow conditions to improve capture yield and purity, and integrating column-based systems with microfluidic platforms and automation to boost throughput, consistency, and reduce operator-dependent variability.

Research has also focused on developing microfluidic-based label-free technologies, such as the integrated ferrohydrodynamic chip or Ni concentrators on a chip, designed for the precise manipulation and separation of rare cells. They aim to improve sensitivity, reduce sample or reagent consumption, and enable rapid, automated assays suitable for point of care use. However, they still face several practical and technical limitations, such as complexity and cost of device fabrication, constraints in geometric and structural design, material instability during prolonged operation, and the limited ability to generate sufficiently high magnetic field gradients. Future research prospects include developing scalable, cost-effective fabrication methods to ensure reproducibility and uniformity of the microfluidic platforms, optimizing device performance with theoretical and computational modeling, and focusing on automation and real-time monitoring to achieve the best performance for clinical applications.

Recent advances in magnetic levitation platforms have significantly enhanced their diagnostic capabilities, enabling the separation and analysis of cells at the single-cell level based on differences in density and magnetic susceptibility.(Karakuzu et al., 2024) These platforms offer rapid, accurate, and user-friendly diagnostics, eliminating the need for targeting ligands and allowing for disease diagnosis through simple visual inspection of cell positions within a microchannel. Although this paper briefly mentions magnetic levitation technologies for detecting in disease diagnostics, prior studies have demonstrated the label-free identification and monitoring of various cell types, including different cancer cell lines, pluripotent stem cell, bacteria, yeast cells, highlighting the broad applicability of magnetic levitation for single-cell analysis across biological domains.(Durmus et al., 2015b), (Puluca et al., 2020) While magnetic levitation platforms show significant promise, they still face limitations that must be addressed before they can be used in clinical applications. To improve the efficiency of magnetic levitation, in addition to employing stronger magnets, researchers have explored increasing the concentration of the paramagnetic medium or using solutions with higher magnetic susceptibility. However, such modifications may impact cell viability and proliferation, necessitating further investigation. (Goreke et al., 2022) Another issue is that magnetic levitation platforms cannot separate cells with very subtle density differences. Future research must focus on integrating image analysis with machine learning and deep learning algorithms to improve this platform’s overall effectiveness and accuracy. Another potential research area is the combination of magnetic levitation with telemedicine platforms for remote patient monitoring. One example is a study done by Baday et al., where they presented a portable, magnetic levitation-based diagnostic platform called i-LEV that integrates cell phone imaging for label-free blood analysis at the point of care, without the need for complex sample preparation or expensive equipment (Baday et al., 2016). The integration with smartphones enables data sharing with healthcare providers, facilitating remote diagnosis and monitoring

Future efforts should focus on enhancing the purity, efficiency, and throughput of label-free technologies, which currently remain suboptimal. This limitation arises from the significantly weaker magnetic forces exerted on unlabeled cells in comparison to those in labeled systems—often differing by several orders of magnitude. To address this, efforts should be directed toward the design and fabrication of devices providing high magnetic fields, and more importantly, high magnetic field gradients within the separation region. Achieving gradients in the order of 1,000 T/m has the potential to substantially increase throughput while simultaneously improving capture efficiency and separation purity in label-free platforms.

6. Conclusions

The effective separation and analysis of rare cells from complex biological fluids, such as blood, are pivotal for advancing diagnostic accuracy and personalized medicine. Magnetophoresis, encompassing both label-based and label-free methods, have emerged as powerful tools in this field. These methods offer advantages over traditional approaches like fluorescence activated cell sorting including reduced costs, simpler operation, and increased portability, making them highly relevant for clinical applications.

In this review, we describe the methodology we employed for selecting and assessing relevant studies in this field, followed by an in-depth analysis of recently published strategies for magnetic-assisted diagnosis. This work covers both label-based and label-free techniques used for targeting and isolating CTCs, fetal cells, and RBCs. The review emphasizes recent advancements in IMNs configurations and magnetic assisted platforms, providing a detailed discussion of their respective benefits and limitations. Label-based techniques, such as Magnetic-Activated Cell Sorting (MACS), utilize IMNs to selectively isolate rare cells with high purity (>80%) and rapid processing (~101⁰ cells/hour). These techniques offer high sensitivity and specificity, facilitating early disease detection and monitoring. The FDA-approved CellSearch system exemplifies the clinical utility of magnetophoresis, enabling real-time cancer monitoring and personalized treatment decisions. On the contrary, label-free approaches, including ferrohydrodynamic cell separation and magnetic levitation, offer promising alternatives by exploiting intrinsic cell properties such as magnetic susceptibility. These methods avoid the need for external labeling, which can potentially preserve cell integrity and function. High-throughput label-free systems have demonstrated remarkable efficiency in isolating CTCs with recovery rates exceeding 80%, offering promising alternatives for non-invasive cancer diagnostics. Additionally, microfluidic-integrated magnetophoresis platforms enable rapid, cost-effective point-of-care diagnostics, further enhancing accessibility to advanced medical testing.

However, optimizing magnetophoresis presents several challenges that must be addressed to ensure their clinical reliability and effectiveness. A critical issue is the synthesis and standardization of magnetic nanoparticles, as inconsistencies in particle size, batch-to-batch variability, and potential cytotoxicity can impact their performance and biocompatibility. Enhancing the sensitivity and resolution of magnetic levitation platforms is essential, particularly for distinguishing materials with minimal differences in density and magnetic susceptibility. Integrating these platforms with advanced computational tools, such as machine learning, could significantly improve accuracy and efficiency in cell separation. However, these devices currently face hurdles related to complex fabrication processes, high costs, and challenges in integrating sensitive detection systems. Future research should prioritize streamlining microfluidic device fabrication, reducing production costs, and developing fully integrated systems that seamlessly combine separation and analysis on a single platform, ultimately advancing their feasibility for clinical and point-of-care applications.

In conclusion, while magnetic-based separation techniques have made remarkable strides in cell analysis and disease diagnosis, ongoing research is crucial to overcoming existing limitations. Standardizing synthesis protocols for magnetic nanoparticles, improving biocompatibility assessments, and advancing microfluidic and label-free technologies will be key to maximizing the potential of these diagnostic tools. By addressing these challenges, we can enhance the reliability and effectiveness of magnetophoresis, paving the way for more accurate and personalized diagnostic approaches in the future.