Nanotentacle‐Structured Magnetic Particles for Efficient Capture of Circulating Tumor Cells

Circulating tumor cells (CTCs) have attracted considerable attention as promising markers for diagnosing and monitoring the cancer status. Despite many technological advances in isolating CTCs, the capture efficiency and purity still remain challenges that limit clinical practice. Here, the construction of “nanotentacle”‐structured magnetic particles using M13‐bacteriophage and their application for the efficient capturing of CTCs is demonstrated. The M13‐bacteriophage to magnetic particles followed by modification with PEG is conjugated, and further tethered monoclonal antibodies against the epidermal receptor 2 (HER2). The use of nanotentacle‐structured magnetic particles results in a high capture purity (>45%) and efficiency (>90%), even for a smaller number of cancer cells (≈25 cells) in whole blood. Furthermore, the cancer cells captured are shown to maintain a viability of greater than 84%. The approach can be effectively used for capturing CTCs with high efficiency and purity for the diagnosis and monitoring of cancer status.     

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.     

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.     

Antibody‐Free Hydrogel with the Synergistic Effect of Cell Imprinting and Boronate Affinity: Toward the Selective Capture and Release of Undamaged Circulating Tumor Cells

The selective and highly efficient capture of circulating tumor cells (CTCs) from blood and their subsequent release without damage are very important for the early diagnosis of tumors and for understanding the mechanism of metastasis. Herein, a universal strategy is proposed for the fabrication of an antibody‐free hydrogel that has a synergistic effect by featuring microinterfaces obtained by cell imprinting and molecular recognition conferred by boronate affinity. With this artificial antibody, highly efficient capture of human hepatocarcinoma SMMC‐7721 cells is achieved: as many as 90.3 ± 1.4% (n = 3) cells are captured when 1 × 10⁵ SMMC‐7721 cells are incubated on a 4.5 cm² hydrogel, and 99% of these captured cells are subsequently released without any loss of proliferation ability. In the presence of 1000 times as many nontarget cells, namely, leukaemia Jurkat cells, the SMMC‐7721 cells can be captured with an enrichment factor as high as 13.5 ± 3.2 (n = 3), demonstrating the superior selectivity of the artificial antibody for the capture of the targeted CTCs. Most importantly, the SMMC‐7721 cells can be successfully captured even when spiked into whole blood, indicating the great promise of this approach for the further molecular characterization of CTCs.     

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.     

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.     

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.     

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.     

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.     

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.     

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     

A Radial Flow Microfluidic Device for Ultra‐High‐Throughput Affinity‐Based Isolation of Circulating Tumor Cells

Circulating tumor cells (CTCs) are believed to play an important role in metastasis, a process responsible for the majority of cancer‐related deaths. But their rarity in the bloodstream makes microfluidic isolation complex and time‐consuming. Additionally the low processing speeds can be a hindrance to obtaining higher yields of CTCs, limiting their potential use as biomarkers for early diagnosis. Here, a high throughput microfluidic technology, the OncoBean Chip, is reported. It employs radial flow that introduces a varying shear profile across the device, enabling efficient cell capture by affinity at high flow rates. The recovery from whole blood is validated with cancer cell lines H1650 and MCF7, achieving a mean efficiency >80% at a throughput of 10 mL h^?1 in contrast to a flow rate of 1 mL h^?1 standardly reported with other microfluidic devices. Cells are recovered with a viability rate of 93% at these high speeds, increasing the ability to use captured CTCs for downstream analysis. Broad clinical application is demonstrated using comparable flow rates from blood specimens obtained from breast, pancreatic, and lung cancer patients. Comparable CTC numbers are recovered in all the samples at the two flow rates, demonstrating the ability of the technology to perform at high throughputs.     

Nanoelectromechanical Chip (NELMEC) Combination of Nanoelectronics and Microfluidics to Diagnose Epithelial and Mesenchymal Circulating Tumor Cells from Leukocytes

An integrated nano‐electromechanical chip (NELMEC) has been developed for the label‐free distinguishing of both epithelial and mesenchymal circulating tumor cells (ECTCs and MCTCs, respectively) from white blood cells (WBCs). This nanoelectronic microfluidic chip fabricated by silicon micromachining can trap large single cells (>12 µm) at the opening of the analysis microchannel arrays. The nature of the captured cells is detected using silicon nanograss (SiNG) electrodes patterned at the entrance of the channels. There is an observable difference between the membrane capacitance of the ECTCs and MCTCs and that of WBCs (measured using SiNG electrodes), which is the key indication for our diagnosis. The NELMEC chip not only solves the problem of the size overlap between CTCs and WBCs but also detects MCTCs without the need for any markers or tagging processes, which has been an important problem in previously reported CTC detection systems. The great conductivity of the gold‐coated SiNG nanocontacts as well as their safe penetration into the membrane of captured cells, facilitate a precise and direct signal extraction to distinguish the type of captured cell. The results achieved from epithelial (MCF‐7) and mesenchymal (MDA‐MB231) breast cancer cells circulated in unprocessed blood suggest the significant applications for these diagnostic abilities of NELMEC.     

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.     

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.     

Targeted Liposome‐Loaded Microbubbles for Cell‐Specific Ultrasound‐Triggered Drug Delivery

One of the main problems in cancer treatment is disease relapse through metastatic colonization, which is caused by circulating tumor cells (CTCs). This work reports on liposome‐loaded microbubbles targeted to N‐cadherin, a cell–cell adhesion molecule expressed by CTCs. It is shown that such microbubbles can indeed bind to N‐cadherin at the surface of HMB2 cells. Interestingly, in a mixture of cells with and without N‐cadherin expression, binding of the liposome‐loaded microbubbles mainly occurs to the N‐cadherin‐expressing cells. Importantly, applying ultrasound results in the intracellular delivery of a model drug (loaded in the liposomes) in the N‐cadherin‐expressing cells only. As described in this paper, such liposome‐loaded microbubbles may find application as theranostics and in devices aimed for the specific killing of CTCs in blood.     

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.     

In Situ Two‐Step Photoreduced SERS Materials for On‐Chip Single‐Molecule Spectroscopy with High Reproducibility

A method is developed to synthesize surface‐enhanced Raman scattering (SERS) materials capable of single‐molecule detection, integrated with a microfluidic system. Using a focused laser, silver nanoparticle aggregates as SERS monitors are fabricated in a microfluidic channel through photochemical reduction. After washing out the monitor, the aggregates are irradiated again by the same laser. This key step leads to full reduction of the residual reactants, which generates numerous small silver nanoparticles on the former nanoaggregates. Consequently, the enhancement ability of the SERS monitor is greatly boosted due to the emergence of new “hot spots.” At the same time, the influence of the notorious “memory effect” in microfluidics is substantially suppressed due to the depletion of surface residues. Taking these advantages, two‐step photoreduced SERS materials are able to detect different types of molecules with the concentration down to 10^?13m . Based on a well‐accepted bianalyte approach, it is proved that the detection limit reaches the single‐molecule level. From a practical point of view, the detection reproducibility at different probing concentrations is also investigated. It is found that the effective single‐molecule SERS measurements can be raised up to ≈50%. This microfluidic SERS with high reproducibility and ultrasensitivity will find promising applications in on‐chip single‐molecule spectroscopy.     

多孔磁性复合微球的制备及其功能化研究进展

与常规磁性微球相比,多孔磁性复合微球具有比表面积大、密度小等特性,因此得到了人们的普遍关注。近几年来,多孔磁性复合微球的制备技术得到了很大发展。目前,基本实现了比表面积可控、孔径和表面化学性质可调。本文详细介绍了多孔磁性复合微球的制备方法,总结了成孔机理和影响孔结构的因素,并对磁性微球表面功能化的方法作了相关概述。     

Folic acid-functionalized magnetic ZnFe2O4 hollow microsphere core/mesoporous silica shell composite particles: Synthesis and application in drug release

A drug delivery system was designed by deliberately combining the useful functions into one entity, which was composed of magnetic ZnFe₂O₄ hollow microsphere as the core, and mesoporous silica with folic acid molecules as the outer shell. Amine groups coated magnetic ZnFe₂O₄ hollow microsphere core/mesoporous silica shell (MZHM-MSS-NH₂) composite particles were first synthesized by a one-pot direct co-condensation method. Subsequently a novel kind of folic acid-functionalized magnetic ZnFe₂O₄ hollow microsphere core/mesoporous silica shell (MZHM-MSS-NHFA) composite particles were synthesized by conjugating folic acid as targeted molecule to MZHM-MSS-NH₂. Ibuprofen, a well-known antiphlogistic drug, was used as a model drug to assess the loading and releasing behavior of the composite microspheres. The results show that the MZHM-MSS-NHFA system has the higher capacity of drug storage and good sustained drug-release property.