Efficient Capture and Simple Quantification of Circulating Tumor Cells Using Quantum Dots and Magnetic Beads

Circulating tumor cells (CTCs) are valuable biomarkers for monitoring the status of cancer patients and drug efficacy. However, the number of CTCs in the blood is extremely low, and the isolation and detection of CTCs with high efficiency and sensitivity remain a challenge. Here, we present an approach to the efficient capturing and simple quantification of CTCs using quantum dots and magnetic beads. Anti‐EpCAM antibody‐conjugated quantum dots are used for the targeting and quantification of CTCs, and quantum‐dot‐attached CTCs are isolated using anti‐IgG‐modified magnetic beads. Our approach is shown to result in a capture efficiency of about 70%–80%, enabling the simple quantification of captured CTCs based on the fluorescence intensity of the quantum dots. The present method can be used effectively in the capturing and simple quantification of CTCs with high efficiency for cancer diagnosis and monitoring.     

Selective Capture and Quick Detection of Targeting Cells with SERS‐Coding Microsphere Suspension Chip

Circulating tumor cells (CTCs) captured from blood fluid represent recurrent cancers and metastatic lesions to monitor the situation of cancers. We develop surface‐enhanced Raman scattering (SERS)‐coding microsphere suspension chip as a new strategy for fast and efficient capture, recovery, and detection of targeting cancer cells. Using HeLa cells as model CTCs, we first utilize folate as a recognition molecule to be immobilized in magnetic composite microspheres for capturing HeLa cells and attaining high capturing efficacy (up to 95%). After capturing cells, the composite microsphere, which utilizes a disulfide bond as crosslinker in the polymer shell and as a spacer for linking folate, can recycle 90% cells within 20 min eluted by glutathion solution. Taking advantage of the SERS with fingerprint features, we characterize captured/recovered cells with the unique signal of report‐molecule 4‐aminothiophenol through introducing the SERS‐coding microsphere suspension chip to CTCs. Finally, the exploratory experiment of sieving cells shows that the magnetic composite microspheres can selectively capture the HeLa cells from samples of mixed cells, indicating that these magnetic composite microspheres have potential in real blood samples for capturing CTCs.     

Size effects of magnetic beads in circulating tumour cells magnetic capture based on streptavidin–biotin complexation

Circulating tumour cells (CTCs) draw significant attention as a promising biomarker for cancer prognosis, status monitoring, and metastasis diagnosis. However, the concentration of CTCs in peripheral blood is usually extremely low, thereby requiring enrichment followed by isolation of CTCs prior to detection. An immunomagnetic separation is a promising tool for CTCs enrichment. In this study, a cost‐effective magnetic separation method, based on streptavidin–biotin complexation, was developed and the effects of magnetic beads’ size in CTCs capture were compared. Magnetic nanobeads which were 25 nm in diameter lead to highest capture efficiency (82.2%) compared with 150 nm magnetic beads and 1 µm microbeads. Based on the streptavidin–biotin system, 25 nm magnetic nanobeads could capture model CTCs over 80% efficiency even at concentrations as low as ∼25 cells/mL that may represent the actual level of CTCs in peripheral blood of cancer patients. Furthermore, the isolated cells remained robust and healthy showing insignificant changes in morphology and behaviour when cultured for 24 h immediately after capture and isolation. The magnetic nanobeads based on streptavidin–biotin complexation showed promise for the easy and efficient capture and isolation of healthy CTCs for further diagnosis and analysis.Inspec keywords: cancer, magnetic separation, nanomedicine, nanomagnetics, proteins, biomagnetism, tumours, cellular biophysics, magnetic particles, molecular biophysics, blood, nanoparticlesOther keywords: streptavidin–biotin complexation, cancer prognosis, peripheral blood, immunomagnetic separation, CTCs capture, streptavidin–biotin system, circulating tumour cells, CTC enrichment, magnetic separation method, magnetic nanobeads, magnetic capture, size 25.0 nm, size 150.0 nm, time 24.0 hour     

Microfluidic-Assisted CTC Isolation and In Situ Monitoring Using Smart Magnetic Microgels

Capturing rare disease-associated biomarkers from body fluids can offer an early-stage diagnosis of different cancers. Circulating tumor cells (CTCs) are one of the major cancer biomarkers that provide insightful information about the cancer metastasis prognosis and disease progression. The most common clinical solutions for quantifying CTCs rely on the immunomagnetic separation of cells in whole blood. Microfluidic systems that perform magnetic particle separation have reported promising outcomes in this context, however, most of them suffer from limited efficiency due to the low magnetic force generated which is insufficient to trap cells in a defined position within microchannels. In this work, a novel method for making soft micromagnet patterns with optimized geometry and magnetic material is introduced. This technology is integrated into a bilayer microfluidic chip to localize an external magnetic field, consequently enhancing the capture efficiency (CE) of cancer cells labeled with the magnetic nano/hybrid microgels that are developed in the previous work. A combined numerical-experimental strategy is implemented to design the microfluidic device and optimize the capturing efficiency and to maximize the throughput. The proposed design enables high CE and purity of target cells and real-time time on-chip monitoring of their behavior. The strategy introduced in this paper offers a simple and low-cost yet robust opportunity for early-stage diagnosis and monitoring of cancer-associated biomarkers.     

Leukocyte‐Repelling Biomimetic Immunomagnetic Nanoplatform for High‐Performance Circulating Tumor Cells Isolation

Downstream studies of circulating tumor cells (CTCs), which may provide indicative evaluation information for therapeutic efficacy, cancer metastases, and cancer prognosis, are seriously hindered by the poor purity of enriched CTCs as large amounts of interfering leukocytes still nonspecifically bind to the isolation platform. In this work, biomimetic immunomagnetic nanoparticles (BIMNs) with the following features are designed: i) the leukocyte membrane camouflage, which could greatly reduce homologous leukocyte interaction and actualize high‐purity CTCs isolation, is easily extracted by graphene nanosheets; ii) facile antibody conjugation can be achieved through the “insertion” of biotinylated lipid molecules into leukocyte‐membrane‐coated nanoparticles and streptavidin conjunction; iii) layer‐by‐layer assembly techniques could integrate high‐magnetization Fe₃O₄ nanoparticles and graphene nanosheets efficiently. Consequently, the resulting BIMNs achieve a capture efficiency above 85.0% and CTCs purity higher than 94.4% from 1 mL blood with 20–200 CTCs after 2 min incubation. Besides, 98.0% of the isolated CTCs remain viable and can be directly cultured in vitro. Moreover, application of the BIMNs to cancer patients' peripheral blood shows good reproducibility (mean relative standard deviation 8.7 ± 5.6%). All results above suggest that the novel biomimetic nanoplatform may serve as a promising tool for CTCs enrichment and detection from clinical samples.     

Magneto‐Controllable Capture and Release of Cancer Cells by Using a Micropillar Device Decorated with Graphite Oxide‐Coated Magnetic Nanoparticles

Aiming to highly efficient capture and analysis of circulating tumor cells, a micropillar device decorated with graphite oxide‐coated magnetic nanoparticles is developed for magneto‐controllable capture and release of cancer cells. Graphite oxide‐coated, Fe₃O₄ magnetic nanoparticles (MNPs) are synthesized by solution mixing and functionalized with a specific antibody, following by the immobilization of such modified MNPs on our designed micropillar device. For the proof‐of‐concept study, a HCT116 colorectal cancer cell line is employed to exam the capture efficiency. Under magnetic field manipulation, the high density packing of antibody‐modified MNPs on the micropillars increases the local concentration of antibody, as well as the topographic interactions between cancer cells and micropillar surfaces. The flow rate and the micropillar geometry are optimized by studying their effects on capture efficiency. Then, a different number of HCT116 cells spiked in two kinds of cell suspension are investigated, yielding capture efficiency >70% in culture medium and >40% in blood sample, respectively. Moreover, the captured HCT116 cells are able to be released from the micropillars with a saturated efficiency of 92.9% upon the removal of applied magnetic field and it is found that 78% of the released cancer cells are viable, making them suitable for subsequent biological analysis.     

Targeting Mucin Protein Enables Rapid and Efficient Ovarian Cancer Cell Capture: Role of Nanoparticle Properties in Efficient Capture and Culture

The development of specific and sensitive immunomagnetic cell separation nanotechnologies is central to enhancing the diagnostic relevance of circulating tumor cells (CTCs) and improving cancer patient outcomes. The limited number of specific biomarkers used to enrich a phenotypically diverse set of CTCs from liquid biopsies has limited CTC yields and purity. The ultra-high molecular weight mucin, mucin16 (MUC16) is shown to physically shield key membrane proteins responsible for activating immune responses against ovarian cancer cells and may interfere with the binding of magnetic nanoparticles to popular immunomagnetic cell capture antigens. MUC16 is expressed in ≈90% of ovarian cancers and is almost universal in High Grade Serous Epithelial Ovarian Cancer. This work demonstrates that cell bound MUC16 is an effective target for rapid immunomagnetic extraction of expressor cells with near quantitative yield, high purity and viability from serum. The results provide a mechanistic insight into the effects of nanoparticle physical properties and immunomagnetic labeling on the efficiency of immunomagnetic cell isolation. The growth of these cells has also been studied after separation, demonstrating that nanoparticle size impacts cell-particle behavior and growth rate. These results present the successful isolation of “masked” CTCs enabling new strategies for the detection of cancer recurrence and select and monitor chemotherapy.     

M13 bacteriophage-activated superparamagnetic beads for affinity separation

The growth of the biopharmaceutical industry has created a demand for new technologies for the purification of genetically engineered proteins.The efficiency of large-scale, high-gradient magnetic fishing could be improved if magnetic particles offering higher binding capacity and magnetization were available. This article describes several strategies for synthesizing microbeads that are composed of a M13 bacteriophage layer assembled on a superparamagnetic core. Chemical cross-linking of the pVIII proteins to a carboxyl-functionalized bead produces highly responsive superparamagnetic particles (SPM) with a side-on oriented, adherent virus monolayer. Also, the genetic manipulation of the pIII proteins with a His(6) peptide sequence allows reversible assembly of the bacteriophage on a nitrilotriacetic-acid-functionalized core in an end-on configuration. These phage-magnetic particles are successfully used to separate antibodies from high-protein concentration solutions in a single step with a >90% purity. The dense magnetic core of these particles makes them five times more responsive to magnetic fields than commercial materials composed of polymer-(iron oxide) composites and a monolayer of phage could produce a 1000 fold higher antibody binding capacity. These new bionanomaterials appear to be well-suited to large-scale high-gradient magnetic fishing separation and promise to be cost effective as a result of the self-assembling and self-replicating properties of genetically engineered M13 bacteriophage.     

Development of a low-cost magnetic microfluidic chip for circulating tumour cell capture

The authors have developed a novel fabrication process for a selective micro-magnetic activated cell sorting (MACS) chip based on ferromagnetic material encapsulated micropillars (FMEMs), which is technically simple and low cost. The FMEM produces a high field gradient to magnetically attract, capture and hold cells on its interface. System test simulations were carried out to predict the efficacy of target capture and verify that the actual magnetic particles behaviour agreed well with model predictions. To determine the ability of the novel microMACS chip to capture circulating tumour cells (CTCs), SW620 human colon cancer cells were used in an in vitro flow model system and were able to be captured with the efficiency of 72.8%. The obvious accumulation of CTCs at a certain location on the chip suggested shear stress events at the pillar boundary may influence efficacy, and should be considered in further optimisation efforts.     

Continuous Flow Deformability‐Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label‐free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label‐free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10⁴‐fold enrichment of target cells relative to leukocytes. In patients with metastatic castration‐resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.     

Engineering Biomimetic Biosensor Using Dual-Targeting Multivalent Aptamer Regulated 3D DNA Walker Enables High-Performance Detection of Heterogeneous Circulating Tumor Cells

The phenotypic heterogeneity of circulating tumor cells (CTCs) and the nonspecific adsorption of background cells impede the effective and sensitive detection of rare CTCs. Although leukocyte membrane coating approach has a good antileukocyte adhesion ability and holds great promise for addressing the challenge of capture purity, its limited specificity and sensitivity prevent its use in the detection of heterogeneous CTCs. To overcome these obstacles, a biomimetic biosensor that integrated dual-targeting multivalent aptamer/walker duplex functionalized biomimetic magnetic beads and an enzyme-powered DNA walker signal amplification strategy is designed. As compared to conventional leukocyte membrane coating, the biomimetic biosensor achieves efficient and high purity enrichment of heterogeneous CTCs with different epithelial cell adhesion molecule (EpCAM) expression while minimizing the interference of leukocytes. Meanwhile, the capture of target cells can trigger the release of walker strands to activate an enzyme-powered DNA walker, resulting in cascade signal amplification and the ultrasensitive and accurate detection of rare heterogeneous CTCs. Importantly, the captured CTCs remained viable and can be recultured in vitro with success. Overall, this work provides a new perspective for the efficient detection of heterogeneous CTCs by biomimetic membrane coating and paves the way for early cancer diagnosis.     

Highly efficient assay of circulating tumor cells by selective sedimentation with a density gradient medium and microfiltration from whole blood

Isolation of circulating tumor cells (CTCs) by size exclusion can yield poor purity and low recovery rates, due to large variations in size of CTCs, which may overlap with leukocytes and render size-based filtration methods unreliable. This report presents a very sensitive, selective, fast, and novel method for isolation and detection of CTCs. Our assay platform consists of three steps: (i) capturing CTCs with anti-EpCAM conjugated microbeads, (ii) removal of unwanted hematologic cells (e.g., leukocytes, erythrocytes, etc.) by selective sedimentation of CTCs within a density gradient medium, and (iii) simple microfiltration to collect these cells. To demonstrate the efficacy of this assay, MCF-7 breast cancer cells (average diameter, 24 μm) and DMS-79 small cell lung cancer cells (average diameter, 10 μm) were used to model CTCs. We investigated the relative sedimentation rates for various cells and/or particles, such as CTCs conjugated with different types of microbeads, leukocytes, and erythrocytes, in order to maximize differences in the physical properties. We observed that greater than 99% of leukocytes in whole blood were effectively removed at an optimal centrifugal force, due to differences in their sedimentation rates, yielding a much purer sample compared to other filter-based methods. We also investigated not only the effect of filtration conditions on recovery rates and sample purity but also the sensitivity of our assay platform. Our results showed a near perfect recovery rate (～99%) for MCF-7 cells and very high recovery rate (～89%) for DMS-79 cells, with minimal amounts of leukocytes present.     

Hyaluronic Acid‐Functionalized Electrospun Polyvinyl Alcohol/Polyethyleneimine Nanofibers for Cancer Cell Capture Applications

Capturing circulating tumor cells (CTCs) with sufficient sensitivity and specificity in vitro is of paramount importance for early cancer diagnosis. Here a facile approach to immobilizing hyaluronic acid (HA) onto electrospun polyvinyl alcohol/polyethyleneimine (PVA/PEI) nanofibers for capturing cancer cells overexpressing CD44 receptors is reported. In this study, electrospun PVA/PEI nanofibers were crosslinked using glutaraldehyde vapor, covalently conjugated with HA via N‐(3‐dimethy‐laminopropyl)‐N′‐ethylcarbodiimide/N‐hydroxysuccinimide coupling reaction, followed by neutralization of the remaining fiber surface PEI amines via acetylation. The formed nanofibers were characterized using different techniques. It is shown that the HA‐modified PVA/PEI nanofibers with a mean diameter of 459.7 nm possess a smooth and uniform fibrous morphology, similar to the PVA/PEI nanofibers without HA modification. The HA‐modified PVA/PEI nanofibers display good cytocompatibility and hemocompatibility as confirmed by cell viability, hemolysis, and anticoagulant assays. Importantly, with the modified HA, the nanofibers exhibit superior capability to capture CD44 receptor‐overexpressing cancer cells. The developed HA‐modified PVA/PEI nanofibers may hold a great promise to be applied for capturing CTCs for cancer diagnosis applications.     

Fast and selective cell isolation from blood sample by microfiber fabric system with vacuum aspiration

Since circulating tumor cells (CTCs) are tumor cells which are found in the blood of cancer patients, CTCs are potential tumor markers, so a rapid isolation of CTCs is desirable for clinical applications. In this paper, a three-dimensional polystyrene (PS) microfiber fabric with vacuum aspiration system was developed for capturing CTCs within a short time. Various microfiber fabrics with different diameters were prepared by the electrospinning method and optimized for contact frequency with cells. Vacuum aspiration utilizing these microfiber fabrics could filter all cells within seconds without mechanical damage. The microfiber fabric with immobilized anti-EpCAM antibodies was able to specifically capture MCF-7 cells that express EpCAM on their surfaces. The specificity of the system was confirmed by monitoring the ability to isolate MCF-7 cells from a mixture containing CCRF-CEM cells that do not express EpCAM. Furthermore, the selective capture ability of the microfiber was retained even when the microfiber was exposed to the whole blood of pigs spiked with MCF-7 cells. The specific cell capture ratio of the vacuum aspiration system utilizing microfiber fabric could be improved by increasing the thickness of the microfiber fabric through electrospinning time.     

Enhanced and Differential Capture of Circulating Tumor Cells from Lung Cancer Patients by Microfluidic Assays Using Aptamer Cocktail

Collecting circulating tumor cells (CTCs) shed from solid tumor through a minimally invasive approach provides an opportunity to solve a long‐standing oncology problem, the real‐time monitoring of tumor state and analysis of tumor heterogeneity. However, efficient capture and detection of CTCs with diverse phenotypes is still challenging. In this work, a microfluidic assay is developed using the rationally‐designed aptamer cocktails with synergistic effect. Enhanced and differential capture of CTCs for nonsmall cell lung cancer (NSCLC) patients is achieved. It is also demonstrated that the overall consideration of CTC counts obtained by multiple aptamer combinations can provide more comprehensive information in treatment monitoring.     

Affinity Versus Label‐Free Isolation of Circulating Tumor Cells: Who Wins?

The study of circulating tumor cells (CTCs) has been made possible by many technological advances in their isolation. Their isolation has seen many fronts, but each technology brings forth a new set of challenges to overcome. Microfluidics has been a key player in the capture of CTCs and their downstream analysis, with the aim of shedding light into their clinical application in cancer and metastasis. Researchers have taken diverging paths to isolate such cells from blood, ranging from affinity‐based isolation targeting surface antigens expressed on CTCs, to label‐free isolation taking advantage of the size differences between CTCs and other blood cells. For both major groups, many microfluidic technologies have reported high sensitivity and specificity for capturing CTCs. However, the question remains as to the superiority among these two isolation techniques, specifically to identify different CTC populations. This review highlights the key aspects of affinity and label‐free microfluidic CTC technologies, and discusses which of these two would be the highest benefactor for the study of CTCs.     

Concentration of Bacillus spores by using silica magnetic particles

Silica particles are mainly used for the concentration of nucleic acid for diagnostic purposes. This is usually done under acidic or chaotropic conditions that will demolish most of the living organisms and prevent the application of other diagnostic tests. Here we describe the development of a method for the capturing and concentration of Bacillus spores using silica magnetic particles to enable fast and sensitive detection. We have shown that capturing various Bacilli spores via silica magnetic particles is limited, with large differences between spore batches (42 +/- 25%). The hydrophobic exosporium layer of spore limits the capture by the hydrophilic silica beads. Partial removal of Bacillus exosporium increases capture efficiency. To increase capturing efficiency without harming the spores' viability, a cationic lipid, didecyldimethylammonium bromide (DDAB), was used as a coat for the negatively charged silica particles. DDAB treatment increased capture efficiency from 42% to more than 90%. Using this method, we were able to capture as few as 100 Bacillus anthracis spores/mL with 90% efficacy. Release of captured spores was achieved by the addition of albumin. The capture and release processes were verified by plating and by flow cytometry using light scatter analysis. The method is simple, efficient, easy to operate, and fast.     

Simple monitoring of cancer cells using nanoparticles

Here we present a new strategy for a simple and fast detection of cancer circulating cells (CTCs) using nanoparticles. The human colon adenocarcinoma cell line (Caco2) was chosen as a model CTC. Similarly to other adenocarcinomas, colon adenocarcinoma cells have a strong expression of EpCAM, and for this reason this glycoprotein was used as the capture target. We combine the capturing capability of anti-EpCAM functionalized magnetic beads (MBs) and the specific labeling through antibody-modified gold nanoparticles (AuNPs), with the sensitivity of the AuNPs-electrocatalyzed hydrogen evolution reaction (HER) detection technique. The fully optimized process was used for the electrochemical detection of Caco2 cells in the presence of monocytes (THP-1), other circulating cells that could interfere in real blood samples. Therefore we obtained a novel and simple in situ-like sensing format that we applied for the rapid quantification of AuNPs-labeled CTCs in the presence of other human cells.     

A Cellular Compatible Chitosan Nanoparticle Surface for Isolation and In Situ Culture of Rare Number CTCs

Circulating tumor cell (CTC) isolation has attracted a great deal of research interest in recent years. However, there are still some challenges, including purity as well as viability of the captured CTCs, resulting from nanoscale structures and inorganic nanomaterials. Here, a chitosan nanoparticle surface is first fabricated by electrospray to provide a cellular compatible interface. The “soft” substrate, further modified by polyethylene glycol (PEG) as an antifouling molecule and DNA aptamer as a specific capture molecule, has a hydrophilic nature and is capable of specific capture of viable rare CTCs from artificial white blood cell (WBC) samples. Furthermore, a subsequent in situ culture strategy based on the developed cellular compatible soft interface is introduced for further purification and proliferation of the captured rare number target cells. The WBCs are weeded out after 2 d, and after a 7 d proliferation nearly 200 MCF‐7 cells are obtained from 7 target cells with more than 90% purity. This work provides a promising strategy for viable isolation and purification of rare CTCs and it has great potential for achieving clinical validity.     

Capturing Cancer: Emerging Microfluidic Technologies for the Capture and Characterization of Circulating Tumor Cells

Circulating tumor cells (CTCs) escape from primary or metastatic lesions and enter into circulation, carrying significant information of cancer progression and metastasis. Capture of CTCs from the bloodstream and the characterization of these cells hold great significance for the detection, characterization, and monitoring of cancer. Despite the urgent need from clinics, it remains a major challenge to capture and retain these rare cells from human blood with high specificity and yield. Recent exciting advances in micro/nanotechnology, microfluidics, and materials science have enable versatile, robust, and efficient cell isolation and processing through the development of new micro/nanoengineered devices and biomaterials. This review provides a summary of recent progress along this direction, with a focus on emerging methods for CTC capture and processing, and their application in cancer research. Furthermore, classical as well as emerging cellular characterization methods are reviewed to reveal the role of CTCs in cancer progression and metastasis, and hypotheses are proposed in regard to the potential emerging research directions most desired in CTC‐related cancer research.