Rational Design of Materials Interface for Efficient Capture of Circulating Tumor Cells

Originating from primary tumors and penetrating into blood circulation, circulating tumor cells (CTCs) play a vital role in understanding the biology of metastasis and have great potential for early cancer diagnosis, prognosis and personalized therapy. By exploiting the specific biophysical and biochemical properties of CTCs, various material interfaces have been developed for the capture and detection of CTCs from blood. However, due to the extremely low number of CTCs in peripheral blood, there exists a need to improve the efficiency and specificity of the CTC capture and detection. In this regard, a critical review of the numerous reports of advanced platforms for highly efficient and selective capture of CTCs, which have been spurred by recent advances in nanotechnology and microfabrication, is essential. This review gives an overview of unique biophysical and biochemical properties of CTCs, followed by a summary of the key material interfaces recently developed for improved CTC capture and detection, with focus on the use of microfluidics, nanostructured substrates, and miniaturized nuclear magnetic resonance‐based systems. Challenges and future perspectives in the design of material interfaces for capture and detection of CTCs in clinical applications are also discussed.


Introduction
Metastasis, the spread of tumor cells from the primary tumor site to vital distant organs through the circulatory system, is directly responsible for most carcinoma-related deaths in cancer patients. [1][2][3] Understanding the metastasis process and REVIEW capture of unique biochemical markers expressed on surface of CTCs. For example, CellSearch system, a typical biochemical interface developed for CTC capture and isolation, is the fi rst FDA-approved system that processes 7.5 mL blood and enriches CTCs by the antibodies of CTCs unique biochemical marker of epithelial cell adhesion molecule (EpCAM) conjugated on magnetic beads, followed by microscopic cell imaging. [ 21 ] Biophysical CTCs capture methods rely on differences in the physical properties of CTCs compared to normal blood cells such as cell size, deformability and density. Filtration and density gradient are two typical conventional biophysical methods for CTC capture and isolation. [ 22,23 ] Specifi cally, fi ltration provides size-based separation of CTCs on the premise that they are larger than normal leukocytes and red blood cells, while density gradient centrifugation utilizes differences in cell density to separate CTCs from blood. Although CTC capture and isolation have been successfully achieved by these systems, the low CTC-capture yield and purity of these systems are matters of concern. [ 24,25 ] Therefore, it is critical and urgent to develop some advanced material interfaces to achieve effi cient capture and subsequent sensitive detection of rare CTCs for advancing biological and clinical cancer studies and applications.
Recently, by exploiting the unique biophysical and biochemical properties of CTCs together with the development of nanotechnologies and advances in microfabrication and microfl uidics, various exquisite material interfaces have been designed for outstanding capture and high-sensitivity detection of rare CTCs ( Figure 1 ). In this review, we summarize recent representative works on the development of advanced material interfaces for CTC capture and detection. First, we will briefl y introduce the known biophysical and biochemical properties of CTCs that can be employed for the design of these material interfaces. Subsequently, we will review the key advanced material interfaces, newly developed for effi cient capture and detection of CTCs that can potentially revolutionize the future healthcare technology in cancer diagnosis and therapy, with focus on microfl uidics, nanostructured substrates, and miniaturized nuclear magnetic resonance-based systems. Lastly, we will present the challenges and future perspectives in the design of innovative materials interface for CTC capture and detection in clinical applications.

Biophysical and Biochemical Properties of CTCs
By presenting the possibility of being exploited to discriminate CTCs from normal blood cells, the biophysical and biomechanical properties of CTCs have gained much attention. [26][27][28] In this section, we fi rst review historical and recent studies of the biophysical properties of CTCs including density, size and deformability. Next, we present studies of the unique biochemical properties of CTCs concentrating on the specifi c surface receptors that can be used for the selective capture and isolation of CTCs from peripheral blood samples.
CTCs were fi rst identifi ed by Ashworth in 1869 when he microscopically inspected the blood from metastatic cancer patients. Due to their similarity to the metastatic cancer cells, CTCs identifi cation were initially done by trained cytologists in term of elongated nuclei and fragmentation of the chromatin based on Papanicolaou's criteria for malignancy. [ 29,30 ] The density of CTCs were then investigated. Seal et al. studied the specifi c gravity of CTCs and leukocytes in density gradient centrifugation, and concluded that the specifi c gravity of CTCs was bigger than leukocytes and the method of density gradient centrifugation appeared to be a potential way for CTCs capture and isolation. [ 31 ] In addition to the biophysical property of density, the tendency of CTCs to form clusters rather than individual cells was reported. [32][33][34] With the development of microscopy and fl uorescent staining technologies, more insight into the biophysical properties of CTCs including size and deformability were provided recently. [ 35,36 ] Several studies in breast cancer and lung adenocarcinoma noted that the size of CTCs was typically larger than blood cells, which has been an important criteria generally used for current CTCs capture and isolation. [ 37,38 ] In addition to size, cell deformability is another important biophysical property of CTCs frequently exploited in current capture and label-free isolation of CTCs. Cell deformability refers to the ability of cells to change shape under a given level of applied stress without rupturing, which is an important biophysical REVIEW (3 of 14) 1500118 wileyonlinelibrary.com property for CTCs to make them survive and transfer in the stressful environment of blood stream. Cell deformability can be indicated by the nuclear-cytoplasmic ratio (NC ratio: the ratio of nuclear area and cell area with the nuclear area subtracted), where cells having larger NC ratio are likely to be less deformable. Meng et al. compared the average NC ratio of CTCs from 36 breast cancer patients with leukocytes, and concluded that CTCs always had larger NC ratio than leukocytes, which was consistent with the cell deformability data in which CTCs was less deformable than leukocytes. [ 39 ] The decreased deformability of CTCs suggests that CTCs are stiffer than leukocytes, which provides an effi cient way for CTCs capture and isolation from leukocytes. [ 40 ] In summary, all the differences of biophysical properties between CTCs and leukocytes in term of density, size, internal structures and deformability demonstrated in these historical and recent studies, could be exploited for the label-free isolation of CTCs.
In addition to the aforementioned biophysical properties, CTCs also express some unique biochemical markers that can be utilized for selective CTC capture and isolation. Among these biochemical markers, EpCAM and human epidermal growth factor receptor 2 (HER2) are two typical biochemical markers frequently used for the isolation and enrichment of CTCs. EpCAM is a transmembrane glycoprotein mediating Ca 2+ -independent cell-cell adhesion in epithelia. [ 41,42 ] EpCAM is found expressed on a great variety of human adenocarcinoma cells, but it is absent in blood cells. [ 43 ] Hence, EpCAM is one expressed CTC-associated biomarkers known, and CTC isolation techniques based on EpCAM antibodies are widely used. [ 44,45 ] The popular CellSearch system, which has been extensively used to capture and isolate CTCs from the blood of patients with cancers of the breast, prostate, and colon, employs a conjugation of EpCAM antibodies to ferrofl uidic beads to enable the capture of CTCs through a magnetic fi eld. [46][47][48] In addition to EpCAM, several studies have reported that HER2 is overexpressed in CTCs of both metastatic and early breast cancer patients, and clinical data has shown that the change of HER2 status from low level expression to high level also occurred along with breast cancer recurrence and progression. [ 49,50 ] On the basis of this fi nding, HER2 is now considered to be a potential CTCs-associated biomarker, and has also been widely used for CTC isolation and enrichment in clinical applications.

Microfl uidics-Based Material Interface for CTCs Capture
Based on the differences in biophysical properties between CTCs and normal blood cells described above, label-free strategy for direct capture and isolation of CTCs can be developed. As a powerful separation approach, microfl uidic technique with small sample-volume requirement, fast processing times, multiplexing capabilities and large surface area-tovolume ratios, offer a good option for label-free CTCs capture and isolation. [ 51,52 ] Recently, with the progress in nanobiotechnology and microfabrication, various microfl uidic devices with rationally designed material interfaces have been developed for effi cient CTCs capture, isolation and enrichment. [53][54][55] These exquisite microfl uidic systems will be briefl y introduced in this section, and their performances on CTCs capture and isolation are summarized in Table 1 .
As a relatively straightforward technique with low cost, size-based microfl uidic fi ltration is one of the fi rst approaches employed for CTCs capture and isolation, based on the fact that CTCs are larger than blood cells such as white blood cells (WBCs) and red blood cells (RBCs). [ 56 ] Several membranebased microfi lters have been developed for size-based microfl uidic capture and isolation of CTCs from peripheral blood samples. [ 57,58 ] Hosokawa et al. developed a microfl uidic device equipped with a nickel microfi lter for size-based selective CTCs capture and isolation ( Figure 2 A). [ 59 ] In this microfl uidic device, the nickel microfi lter composed of 100×100 holes with the diameters between 8 and 11 µm was integrated between two PDMS funnels. Based on this microfl uidic device, effi cient isolation of CTCs from peripheral blood samples was obtained with high effi ciency of greater than 80%. One advantage of this microfl uidic device over conventional immunomagnetic CTCs separation platforms is its unique ability for effi cient isolation of EpCAM-negative CTCs. Moreover, approximately 98% of captured CTCs were found to be viable after fl uorescent staining and washing processes. In another report, Vona et al. developed a polycarbonate microfi lter-based microfl uidic device for size fi ltration of CTCs. The polycarbonate microfi lter presented holes with diameter of 8 µm, and CTCs were effi ciently trapped by this microfl ter while RBCs and WBCs with smaller sizes www.MaterialsViews.com www.advancedscience.com Figure 1. By exploiting the unique biophysical and biochemical properties of CTCs, various exquisite platforms for CTCs capture and detection have been designed, which mainly involve the microfl uidic-, nanostructured substrates-, and µNMR-based systems. Some representative examples are exhibited here. Reproduced with permission. [ 59 ] Copyright 2010, American Chemical Society. Reproduced with permission. [ 66 ] Copyright 2010, National Academy of Sciences. Reproduced with permission. [ 84 ] Copyright 2012, Royal Society of Chemistry. Reproduced with permission. [ 94,101,122 ] passed through it. Furthermore, this microfl uidic system could run 12 samples in parallel and performed further identifi cation and characterization of CTCs. In addition to above mentioned 2D microfi lter-based microfl uidic devices, Zheng et al. recently fabricated a microfl uidic system based on a microfi lter with 3D pore structure for CTC isolation. [ 61 ] The 3D microfi lter was composed of two 10 µm thick Parylene C membranes separated by a 6.5 µm gap. Since the microholes fabricated on the top membrane were misaligned with that fabricated on the bottom membrane, large CTCs were effi ciently trapped in this fi lter and fi nally, CTCs isolation from peripheral blood samples was successfully achieved based on this 3D microfi lter-based microfl uidic platform. Moreover, a comparative study between the 2D and 3D microfi lters was carried out, and it was found that the 2D microfi lter might damage CTCs during the microfi ltration process but the 3D microfi lter could resolve this problem.
Additionally, microfl uidic devices with functionalized microchannels have been developed for CTCs capture and isolation. [62][63][64] Launiere et al. fabricated a microfl uidic system with channels modifi ed by alternating patterned biomimetic proteins (EpCAM antibody and E-selectin) to increase target CTCs capture while reducing leukocyte's non-specifi c adhesion by up to 82%. [ 62 ] In addition to microfl uidic platforms with barely immunoaffi nity-based strategy for CTCs capture, microfl uidic www.MaterialsViews.com www.advancedscience.com Figure 2. Evolution of the CTCs capture and enrichment methods based on microfl uidics-based material interfaces. A) Microcavity fi lter. Reproduced with permission. [ 59 ] Copyright 2010, American Chemical Society. B) CTC-chip. Reproduced with permission. [ 17 ] Copyright 2007, Macmillan Publishers Ltd. C) Herringbone-chip. Reproduced with permission. [ 66 ] Copyright 2010, National Academy of Sciences. D) Arc-shaped trap. Reproduced with permission. [ 80 ] Copyright 2010, Elsevier. E) Spiral microfl uidic channel. Reproduced with permission. [ 84 ] Copyright 2012, Royal Society of Chemistry.  [ 65 ] This platform consisting of 59 000 micropillars, which could improve the interactions between CTCs and the aptamers, achieved effi cient CTCs capture with effi ciency of 95% from non-processed whole blood samples. Nagrath et al. developed a unique microfl uidic platform with EpCAM antibody-coated microposts array for effi cient and selective CTCs capture and isolation. [ 17 ] Based on the selective interactions between target CTCs and the EpCAM antibody-coated microposts, viable CTCs isolation from peripheral whole blood samples was achieved by this platform under precisely controlled laminar fl ow conditions. It was found that this platform successfully identifi ed CTCs from peripheral whole blood samples of patients with different cancers with high sensitivity of 99% (Figure 2 B). In addition to the functionalized micropost array-based microfl uidic platform, the same group also reported a herringbone-based high-throughput microfl uidic mixing device (Herringbone-Chip) for enhanced CTCs capture and isolation (Figure 2 C). The Herringbone-Chip consisted eight microchannels with patterned herringbone structures designed to generate microvortices and provide passive mixing of blood cells to enhance the interactions between CTCs and EpCAM antibody-coated chip surface. Consequently, CTCs capture with effi ciency of 91.8% was obtained based on this herringbone-Chip in the blood samples prepared by spiking defi ned numbers of cancer cells into blood, as well as clinical blood samples from patients with metastatic disease, thereby indicating its great potential in clinical settings. [ 66 ] Liu et al. developed a microfl uidic devices with EpCAM antibody-functionalized deterministic lateral displacement (DLD) chamber composed of triangular micropost arrays for CTCs capture. [ 67 ] Based on the combination of microfl uidic DLD array and high affi nity-based capture approach, effi cient CTCs capture with 90% effi ciency was obtained from spiked blood samples at low cell concentration (10 2 cells mL −1 ). Kamande et al. reported a modular microfl uidic system containing three different functional regions by which isolation, enumeration, and phenotyping of CTCs could be fi nished in one device. [ 68 ] By combing the immunomagnetic separation strategy and microfl uidic technique, Immunomagnetic-based microfl uidic systems have also been developed for effi cient CTCs capture and isolation. [69][70][71][72][73][74][75][76] Hoshino et al. developed an immunomagnetic-based microfl uidic device for CTCs capture. [ 69 ] In this work, blood samples were fi rstly labelled with magnetic nanoparticles functionalized by EpCAM antibodies, and target CTCs were then effi ciently captured with high effi ciency of 90% when the blood samples fl owed through the microfl uidic channel closely above arrayed magnets. Huang et al. also reported an immunomagnetic-based microfl uidic system for CTCs isolation from blood samples. [ 70 ] This microfl uidic system was operated in a fl ip-fl op mode in order to reduce the stagnation and non-specifi c adhesion of normal blood cells on microfl uidic surface in the process of CTCs capture, and high capture effi ciency of 90% was fi nally achieved based on this platform. The same group also developed a versatile immunomagnetic nanocarrier-based microfl uidic platform for capturing CTCs in whole blood. [ 71 ] In this work, CTCs were selectively targeted by EpCAM antibody-functionalized magnetic nanocarriers and isolated from whole blood samples by magnetic force in a microfl uidic chamber with capture effi ciency greater than 90%. Chen et al. fabricated a graphite-coated magnetic nanoparticles microarray chip for CTCs capture and isolation. [ 72 ] The graphite-coated magnetic nanoparticles with good biocompatibility and stability were synthesized by using the chemical vapor deposition, and the graphite modifi cation on its surface provided functional groups for subsequent antibody labelling to achieve specifi cally CTCs recognition. Based on this graphitecoated magnetic nanoparticles microarray chip, effi cient CTCs capture from spiked blood samples was successfully achieved even at very low cell concentrations. Similarly, Yu et al. developed a microfl uidic system with micropillar array decorated with graphite oxide-coated and antibody-functionalized magnetic nanoparticles for CTCs capture and isolation. [ 73 ] Under magnetic fi eld manipulation, the decoration of functionalized magnetic nanoparticles on the micropillars increased the interactions between target CTCs and micropillar surface, and successful CTCs capture from two spiked media was achieved with capture effi ciency greater than 70% in culture medium and greater than 40% in blood sample. In another report, Issadore et al. developed a microfl uidic chip-based micro-Hall detector to capture immunomagnetic nanoparticle-tagged CTCs from whole blood sample with high effi ciency and high-throughput ability. [ 74 ] High deformability is a distinctive biomechanical property for cells circulating in the peripheral blood, especially for CTCs with larger size than normal blood cells in order to rapidly go through capillaries with small diameters of 6-8 µm and successfully metastasize. [ 77,78 ] Atomic force microscopy (AFM)-based single cell stiffness study for different cancer cells including lung, breast and pancreatic, have shown that malignancy increase cell deformability at the single cell level although CTC are still more stiffer than blood cells. [ 40 ] Therefore, in addition to size, the unique deformability of CTCs is also a factor that can be used for selective capture and isolation of CTCs. Based on the fact that CTCs are always larger and stiffer than normal blood cells, our group developed a microfl uidic device equipped with an array of traps for CTCs capture and isolation from peripheral blood samples. [ 79,80 ] For the structure of trap array, each trap was composed of three pillars with a diameter of 3-4 µm and was arranged in an arc shape with 5 µm distance between pillars. In the process of CTCs capture and isolation, small sized RBCs and WBCs with higher deformability could pass through the 5 µm gaps, while larger CTCs were stuck in the arc-shaped traps, achieving highly effi cient CTCs capture and isolation (Figure 2 D). Furthermore, a pre-fi lter with 20 µm gap was mounted to prevent larger clumps and debris from clogging up the cell trap area. Highly isolated CTCs can be fi nally collected from this microfl uidic-based material interface for downstream applications, such as immunological staining and molecular analysis.
In a straight microfl uidic channel, fl uid shear can generate lateral forces to cause transverse migration of particles. [ 81,82 ] While in a spiral microfl uidic channel, an inertial focusing of particle according to its size can be observed due to combination of shear induced life force and Dean drag force, which www.MaterialsViews.com www.advancedscience.com has been used for size separation of particles, giving some illumination for CTCs isolation by using a spiral microfl uidic channel. Separation of CTCs in a spiral channel with rectangular cross-section has been reported. [ 83 ] Recently, a novel spiral microfl uidic device with trapezoidal cross-section was developed for rapid and effi cient label-free isolation of CTCs from clinically blood samples, by utilizing the inherent Dean vortex fl ow and inertial lift forces present in the spiral microfl uidic channel (Figure 2 E). [ 84 ] Compared to conventional spiral microfl uidic devices with rectangular cross-section, the position of Dean vortex core in spiral microchannel with trapezoidal crosssection can be altered by which larger CTCs will focus and be collected at the inner channel wall outlet while smaller hematologic cells will focus and be removed at the outer wall outlet, thus achieving effi cient CTCs isolation and enrichment. Based on this platform, high CTC capture effi ciency of greater than 80% were successfully achieved within 8 min from both spiked cancer cells blood samples and clinical peripheral blood samples from patients with advanced stage metastatic breast and lung cancers, providing a powerful tool for CTCs capture and isolation.

Nanostructured Substrates-Based Materials Interface for CTC Capture
In tissue engineering and regenerative medicine, nanostructured substrates have been widely employed to mimic the natural extracellular matrix (ECM) and basement membrane. [85][86][87] These substrates can promote cell attachment due to enhanced local topographic interactions between nanostructures and nanoscale components of the cellular surface such as microvilli and fi lopodia, thereby assisting the capture and isolation of CTCs. [ 88,89 ] Furthermore, nanostructured substrates can provide more surface area for immobilization of CTC affi nity molecules. [ 90,91 ] Hence, nanostructured substrates can be combined with the affi nity interactions-based CTC capture strategy, which can further improve CTC capture effi ciency and emerge as a promising platform for isolation, and enrichment of CTCs. In this section, different types of nanostructured substratesbased platforms for CTCs capture and isolation available will be briefl y introduced including nanowires, nanopillars, nanodots, nanofi bers, nanosheets, nanotubes and nanopores, and their performances on CTCs capture and isolation summarized in Table 2 .
Inspired by the surface components of cells, the nanowire and nanopillars-based substrates have been designed and utilized to make use of the surface adhesion of the cells and aid the capture of CTCs in blood samples. For example, Wang et al. fi rstly utilized anti-EpCAM-coated Si nanopillars (SiNPs) substrates to identify and capture CTCs ( Figure 3 A). Using a wet chemical etching approach, densely packed nanopillars of 100-200 nm in diameter were prepared on silicon wafers; additionally, the length of these nanopillars could be easily controlled by altering the etching times. To test the cell capture effi ciency of the SiNPs, cell suspension solution of MCF7 cells (an EpCAM-positive cell line) was introduced for 1 h into the SiNPs and also fl at silicon substrates. It was found that more cells were captured on SiNPs (45-65%) than on fl at silicon substrates (4-14%), suggesting that nanopillars are responsible for enhanced cell capture. The performance of SiNPs on CTC capture and isolation was tested in the artifi cal CTCs blood samples prepared by spiking blood with different densities of tumor cells, and improved capture effi ciency of CTCs (40%) was obtained by the SiNPs platforms comparted to some commercially available technologies. [ 92 ] Similar to SiNPs, quartz nanowires (QNWs) was also fabricatied and employed for CTCs capture and quantifi cation in the spiked blood samples to investigate its potential in clinical use. [ 93 ] By increasing the contact frequency between cell and nanopillar substrate, even higher capture effi ciency of CTCs could be further achieved. For instance, Wang et al. integrated SiNPs into a microfl uidic device with serpentine chaotic micromixers, obtaining a nearly 100% capture effi ciency (Figure 3 B). To test the performance of this integrated platform for CTC capture, a series of CTC samples was fi rstly prepared by spiking three kinds of solutions (whole blood, lysed blood, and PBS buffer) with cancer cell lines of MCF7, T24 and PC3, respectively. Under the optimal conditions of fl ow rate, more than 95% of capture effi ciency of target cancer cells was found in all CTCs samples mentioned above by this integrated platform, providing an effi cient way for isolation of CTCs and early diagnosis of cancer metastasis. [ 94 ] Nanodot and nanosheet substrates have also been demonstrated to show effi cient CTCs capture ability. The dot size and density were controlled by the voltage applied and could be easily reproduced and tuned. Five tumor cell lines of interest were examined and they were either overexpressed with EpCAM antigens or without EpCAM antigens on their cell membranes. Although the aspect ratios of nanodots were small, the effi ciency of specifi c cell capture by anti-EpCAM conjugated to the nanodots, was enhanced by four to fi ve times in comparison with smooth fi lms (Figure 3 C). [ 95 ] The enhancement is most likely due to a synergistic effect from ligand-receptor interaction, and nanostructure matching of tumor cells and nanodot substrate. In addition to nanodot, nanosheet substrates have also been employed for effi cient CTCs capture and isolation. Yoon et al. fabricated a graphene oxide nanosheet substrate-based device and used it for CTCs capture from blood samples. [ 96 ] After functionalized by CTCs-selective antibodies, this nanosheet substate-based device exhibited ability for CTCs capture with effi ciency of 73% from blood samples of pancreatic, breast and lung cancer patients at low cell concentrations (3-5 cells mL −1 ). Inspired by ECM scaffolds, nanofi ber-based substrates have been fabricated and well developed for their effi cient CTCs capture effi ciency. Different materials such as TiO 2 , and poly(lacticco-glycolic acid) (PLGA) can be electrospun to form desired nanofi bers with controllable diameters and lengths. Zhang et al. fabricated TiO 2 nanofi bers of 100-300 nm diameter from a spun composite of titanium n-butoxide and polyvinyl pyrrolidone (Figure 3 D). By coating anti-EpCAM onto the surface of nanofi bers, functionalized platform for CTCs capture was prepared. Using these nanofi bers deposited substrates, cancer cells from artifi cial CTCs blood samples, as well as from whole blood samples of colorectal and gastric cancer patients were reliably captured. [ 97 ] In another report, Hou et al. developed a PLGAnanofi ber embedded chip (PN-nanovelcro chip) which not only captured CTCs with high effi ciency, but also enabled highly specifi c isolation of single melanoma cell immobilized on the nanosubstrate. [ 98 ] The PN-nanovelcro chip was composed of an overlaid PDMS chaotic mixter and a transparent PN-nanovelcro substrate fabricated by electrospining PLGA nanofi bers onto a commercial laser microdissection (LMD) slide and functionalized by a melanoma-specifi c antibody. Based on the enhanced local interaction between cell and PLGA nanfi bers, target melanoma cells were effi ciently captured, and single cell isolation was subsequently isolated by using the highly accurate LMD www.MaterialsViews.com www.advancedscience.com Figure 3. Effi cient capture and enrichment of CTCs by using nanostructured substrates-based material interface. A) Nanopillar substrates. Reproduced with permission. [ 92 ] B) Integrated chaotic micromixer-nanopillar substrates. Reproduced with permission. [ 94 ] C) Nanodot. Reproduced with permission. [ 95 ] D) Nanofi ber. Reproduced with permission. [ 97 ] E) Carbon nanotubes. Reproduced with permission. [ 99 ] F) Nanopore-based 3D graphene foam. Reproduced with permission. [ 101 ] technique. In order to specifi cally identify melanoma cells captured on the PLGA nanofi bers, a four-color immunocytochemistry method was also developed in the PN-nanovelcro chip system.
Nanotubes and nanopores have also been reported to have great potential for CTCs capture and isolation. For example, functionalized multiwalled carbon nanotubes (MWCNTs) fi lms have been successfully used for K562 cells (leukemia cells) capture and electrochemical sensing (Figure 3 E). [ 99 ] They prepared the fi lms by covalent coupling between -NH 2 groups in 3-aminophenylboronic acid (APBA) and -COOH groups in acid-oxidized MWCNTs. Due to the high affi nity interacions between the boronic acid groups of APBA and the carbohydrate on cell surface, the K562 cells could be effi ciently captured by the APBA-functionalized MWCNTs fi lms. Compared to bare APBA fi lms, the functionalized MWCNTs one not only exhibited more boronic acid groups for K562 cell recognition, but also provided enhanced local cell-MWCNTs interactions, which improve K562 cells' adhesion on its surface. Furthermore, the high electrical conductivity of MWCNTs maked the APBA-MWCNTs fi lm a good electrode for subsequent cell electrochemical sensing, presenting a promising way for effi cient capture and highly sensitive electrochemical detection of CTCs. In another report, King et al. explored a method to more effi ciently capture leukemic and epithelial cancer cells from fl ow by altering the nanoscale topography of the inner surface of P-selectin-coated microtubes. [ 100 ] In this work, halloysite nanotubes were naturally attached to the inner surface of microtubes to alter their nanoscal topography via a monolayer of poly-L-lysine. It was found that the capture effi ciency of leukemic cells could be increased by halloysite nanotube coatings and mainly affected by halloysite content and selectin density, making the functionalized microtubes with nanoscale topography a promising platform for enhanced CTCs capture and isolation. In addition to nanotubes, nanopores also open up a new opportunity in CTCs capture and diagnosis. In a recent report, we reported a 3D hierarchical graphene platform that combines microporosity from reduced graphene oxide foam with anti-EpCAM coated ZnO nanorod array (Figure 3 F). [ 101 ] The advantage of this novel composite structure stems from its high density of ZnO nanorods, which increases cell-substrate contact frequency, as well as its microporosity, which lets through normal RBCs but specifi cally captures CTCs due to the introduction of EpCAM antibodies. When thickness of the foam reached 5 mm, the cellcapture yield was more than 80%, indicating its potential CTCs capture capability for clinical blood samples.

Miniature Nuclear Magnetic Resonance System-Based Materials Interface for CTCs Capture and Detection
As a novel sensing technology, micro-nuclear magnetic resonance ( µ NMR) exploits magnetic resonance technology to detect target labelled with immunospecifi c magnetic nanoparticles (MNPs), showing great potential in rapid and highly sensitive biodetection. [ 102 ] The typical MNPs used in µ NMR are superparamagnetic and have small size (tens of nm), which is different from the conventional magnetic nanoparticles used in immunoseparation. The mechanism of µ NMR-based sensing technique is based on the phenomenon that MNP-labeled targets exhibit faster relaxation of NMR signals due to local magnetic fi elds created by MNPs. [ 103 ] By systematically optimization of nanoagents, MNP-target conjugation method, and NMR detectors, several exquisite µ NMR-based platform have been developed for rapid and sensitive detection of biomolecules including nucleic acids, proteins, bacteria, and tumor cells. [104][105][106][107][108][109] Compared to conventional biosensing methods, µ NMR-based technique do not need sample purifi cation procedures and can simultaneously achieve target capture and detection, gaining much attention in the fi elds of CTCs capture and detection. This section will briefl y introduce recent developments of µ NMR-based biosening systems and their potential applications in CTCs capture and detection.
Based on the " T2 -shortening" effect of MNPs in NMR measurements, the detection of CTCs labelled with MNP can be achieved. In NMR measurements, MNPs can produce local magnetic dipole fi elds with strong spatial dependence, and subsequent destroy the coherence in the spin-spin relaxation of water protons. Therefore, target labelled with MNP will show shorter transverse relaxation time in NMR measurements, namely the phenomenon of " T2 -shortening" effect, compared to target without MNPs label, making detection of CTCs possible ( Figure 4 A). [ 102 ] For µ NMR-based CTCs detection system, engineering MNPs for high transverse relaxivity and effi cient MNP labeling on cells are two important issues which need to be addressed for highly sensitive µ NMR sensing. [ 110 ] For the fi rst issue, elemental iron (Fe) exhibiting the highest saturation magnetization and low magnetocrystalline anisotropy among ferromagnetic crystals, may be a good candidate of constituent material for MNPs and it is possible to synthesize superparamagnetic Fe-MNPs with high transverse relaxivity. Recently Yoon et al. synthesized a new type of hybrid Fe-MNP with high magnetic moments and transverse relaxivity. [ 111 ] This hybrid particle composed of an elemental Fe core and a protective ferrite shell, showed high transverse relaxivity and stable magnetic properties against oxidation. In addition to the synthesis of MNPs with high transverse relaxivity, strategies for effi cient cell MNP-labeling are also needed for µ NMR-based CTC capture and detection. Recently, a novel labelling strategy for target-MNPs constructs preparation called BOND (Bioorthogonal nanoparticle detection) was developed by Lee et al. [ 112 ] Based on the reaction between tetrazine (Tz) and trans-cyclooctene (TCO), namely the Diels-Alder cycloaddition, BOND can rapidly achieve the covalent binding of MNP to biological targets at room temperature without catalyst (Figure 4 B). BOND chemistry has been employed for cell MNPs labelling using a twostep approach: cell labelling with TCO-modifi ed antibodies, and the subsequent covalent binding between cell-antibodies-TCO and Tz-loaded MNPs. Since one antibody can be modifi ed by multiple TCO tags without loss of its affi nity, multiple attachment of Tz-MNPs to cells can be subsequently achieved by using the antibodies as scaffolds. Therefore, compared to the method for cell-MNPs preparation directly using MNP-antibody conjugates, the two-step BOND strategy can effi ciently amplify MNP-binding to cells, and then will amplify NMR signals and ultimately enhanced the detection sensitivity, thus showing great potential in µ NMR-based CTC capture and detection. Till now, different models of µNMR devices with miniaturized system for CTCs capture and point-of-care detection have been developed. The miniaturization of µNMR system endows the following advantages: 1) improving detection sensitivity by reducing sample volumes and increasing the concentrations of targets; and 2) generating stronger radio-frequency NMR magnetic fi elds due to the use of smaller magnets in miniaturized system. Figure 4 C shows a typical miniaturized µNMR device developed for CTCs capture and detection, which included four main parts: microcoils, microfl uidic network, custom-designed NMR electronic and a portable small permanent magnet. On the bottom of this µNMR device, eight planar microcoils with volume of 10 µL were arranged into a 2 × 4 array format to achieve parallel CTCs detection. A microfl uidic network was implemented on the top of the microcoils to enable sample handling and distribution. In addition, to compensate for the www.MaterialsViews.com www.advancedscience.com Figure 4. A) Principle of CTC detection based on µ NMR system. CTCs tagged with MNPs can accelerate the transverse relaxation of water protons. Compared to the non-tagged samples (left), the NMR signal will decay faster in time domain (right), providing a sensing mechanism. Reproduced with permission. [ 102 ] Copyright 2008, Macmillan Publishers Ltd. B) Bioorthogonal nanoparticle detection (BOND). The method is based on the Diels-Alder cycloaddition between trans-cyclooctene (TCO) and tetrazine (Tz). Cells are pre-labeled with TCO-antibodies and targeted with Tz-MNPs. The antibody provides sites for multiple MNP binding. Reproduced with permission. [ 112 ] Copyright 2010, Macmillan Publishers Ltd. C) Typical example of the µ NMR system. This system consists of an array of microcoils for NMR detection, microfl uidic channels for sample handling, embedded NMR electronics, and a permanent magnet. Reproduced with permission. [ 102 ] Copyright 2008, Macmillan Publishers Ltd. D) CTC deteciton performance comparison between µNMR and CellSearch system. Reproduced with permission. [ 114 ] Copyright 2012, Neoplasia Press.
inhomogeneity of magnetic fi eld generated by the small permanent magnet, a NMR electronic was customarily designed and implemented to allow for spinecho measurement. Appreciating the rarity of CTCs in blood sample and the heterogeneity of CTCs surface biomarkers, quad-µNMR platform with a quad biomarker "cocktail" (MUC-1, EGFR, HER2, and EpCAM) for optimal signal and detection was developed for CTCs isolation and detection. [ 113 ] In this cocktail assay, CTCs were simultaneously targeted with TCO-modifi ed MUC-1, EGFR, HER2, and EpCAM antibodies and subsequently incubated with Tz-MNPs to get the quad NMR probes of CTC-MNPs. For the performance of this quad-µNMR platform, it was found that an average recovery rate of 38% across the various cell concentrations (200, 100, 50, and 25 spiked cells) tested was obtained, which was higher than the CellSearch system only with an average recovery rate of 9.1%, ultimately leading to higher CTCs detection sensitivity ( Figure 5 D). Compared to CellSearch system, the quad-µNMR platform demonstrated 400% fold higher CTCs detection sensitivity, showing great potential in isolation and detection of CTCs with low surface biomarkers expressing cell line, such as the MDA-MB-436 with known EMT behavior. Furthermore, CTCs isolation and detection from peripheral blood samples collected from 15 patients with ovarian cancer were successfully and sensitively achieved by using the quad-µNMR platform, indicating its potential in clinical applications. [ 114 ] In summary, the Quad-µNMR platform expands the range of CTCs isolation and detection conditions, making it not limited to the case of higher CTCs concentrations such as stage IV, progressive disease, or in patients not pursuing active therapy.

Approaches for CTC Detection and Identifi cation
Once CTCs are captured and enriched, subsequent detection and identifi cation are needed to investigate their origin and genetic profi le from which more valuable insight into the biology of metastasis can be obtained. [ 115,116 ] In µNMR-based platforms, CTCs detection can be easily achieved by analyzing the NMR signals of MNP-labelled target tumor cells without prerequisite isolation and enrichment processes. Hence, this section focuses on the approaches of CTCs detection and identifi cation used in microfl uidic-and nanostructured substratesbased platforms, which mainly involve immunological and molecular methods.
Most CTCs immunological identifi cation assays use different fl uorescent dyes to simultaneously stain cytokeratins (positive marker for epithelial tumor cells) and leukocyte antigen CD45 (exclusion marker). The cell staining process is always carried out in situ in microfl uidic-and nanostructured substrates-based platforms. For example, in size fi ltration-based microfl uidic system with microcavity arrays, two fl uorescent immunological probes of FITC-labelled anti-CD45 antibody and www.MaterialsViews.com www.advancedscience.com Figure 5. Strategies for controllable CTC release from nanowire substrates. A) Enzymatic treatment. Reproduced with permission. [ 121 ] B) temperature stimulation. Reproduced with permission. [ 122 ] C) pH and glucose stimulation. Reproduced with permission. [ 123 ] Copyright 2013, American Chemical Society. (11 of 14) 1500118 wileyonlinelibrary.com PE-labelled anti-EpCAM antibody were employed to detect and identify CTCs captured on the microcavity arrays. [ 59 ] Similarly, in a recent study, CTCs captured on the aptamer-functionalized SiNWs substrates were distinguished from non-specifi cally trapped WBCs by using a three-color immunological method based on FITC-labeled anti-EpCAM, Cy5-labeled anti-CD45, and DAPI nuclear staining. [ 117 ] Most CTCs molecular identifi cation methods use DNA testing techniques such as polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) to analyze the specifi c DNA or mRNA of CTCs enriched. [ 117,118 ] PCR-based analysis technique are the most widely used molecular method for CTCs detection and identifi cation. For example, Devriese et al. used PCR-based technique to analyze a panel of gene marker of CTCs including cytokeratin 7, cytokeratin 19, human epithelial glycoprotein and fi bronectin 1 for selective identifi cation and detection of CTCs in non-small lung cancer, and achieved sensitivity of 46% and a specifi city of 93% in 46 cancer patient. [ 119 ] In another study, Hoe et al. used PCR to do the single CTC genotyping for a key melanoma drug target mutation after capturing CTCs using a nanofi ber-embedded microchip. [ 98 ] For approaches of CTCs detection and identifi cation, there are still two factors needed to be taken into consideration. Firstly, heterogeneity among CTCs is a problem that can not be ignored for CTCs detection and identifi cation, which makes cell-to-cell variations occur in same cancers and only a very small fraction of CTCs that may eventually acquire the ability to seed the metastatic tumor. Hence, how to characterize molecular, phenotypic and functional difference of CTCs at the single-cell level is a critical problem that need to be reviewed. Lee et al. reported a laser scanning cytometry-based method for CTCs detection and identifi cation, by which automated and rapid characterization of physical and functional cellular properties such as size, shape and signaling proteins of CTC at the single-cell level was quantitatively achieved. [ 93 ] In another report, by combining the PLGA nanofi ber substrate with the laser microdissection (LMD) technique, an exquisite platform for CTCs capture and detection was successfully developed by Tseng group. [ 98 ] In this work, based on an LMD microscope, captured CTCs could be cut out and harvested at the single-cell level, making subsequent singlecell molecular analysis possible. In addtion to the problem of CTCs heterogeneity, controllable release strategy of CTCs after capture are also needed for subsequent detection and identification of CTCs. For microfl uidic CTCs capture platforms, magnetic-based release strategy is the main method widely used in CTCs molecular identifi cation process. [70][71][72][73]120 ] For example, in a recent study, Yu et al. developed a microfl uidic platform with micropillar array decorated with magnetic nanoparticles for CTCs capture with effi ciency of greater than 70% when the magnetic fi eld was applied, and the captured CTCs could be released with high effi ciency of 92.9% upon the removal of applied magnetic fi eld. [ 73 ] Moreover, it was found that 78% of the released CTCs was viable, laying a solid foundation for subsequent molecular analysis. For nanostructured substrates-based CTCs capture platforms, different CTCs release strategies have been reported, which can be divided into three categories: enzymatic treatment, temperature, and pH and glucose dual stimulation. Detailed explanation for the three strategies will be given below. Enzymatic strategy for controllable CTC release after capture was fi rstly demonstrated for the SiNWs-based CTCs capture platform by Shen et al. (Figure 5 A). In their work, by modifi ed the SiNWs substrates with CTC selective DNA aptamers generated via Cell-SELEX process, a new integrated SiNWsmicrofl uidic chaotic mixture-based CTCs capture platform was fabricated. This aptamers-functionalized platform could not only achieve effi cient CTCs isolation from blood with improved capture effi ciency compared to the conventional EpCAM-functionalized platform, but also realize controllable CTCs release after capture by nuclease treatment. [ 121 ] In another report, Hou et al. developed an exquisite platform with CTC capture and ondemand release ability based on thermally responsive Poly( Nisopropylacrylamide) (PNIPAAm) brushes-modifi ed SiNWs substrate (Figure 5 B). This platform exhibited superior performances in capturing cancer cells with high effi ciency at 37 °C, and releasing the captured cancer cells with great viability and retained functionality at 4 °C. [ 122 ] Recently, Jiang et al. developed a pH and glucose-responsive strategy for CTCs release after capture based on poly(acrylamidophenylboronic acid) (polyAAPBA) brush-grafted aligned SiNWs substrate (Figure 5 C). By precisely controlling pH and the glucose concentration in CTCs samples, reversible capture and release of CTCs could be successfully achieved with dual-responsive performance. Specifi cally, the polyAAPBA-grafted SiNWs substrate changed its state from cell-adhesive to cell-repulsive with the increase of pH from 6.8 to 7.8 in the presence of 70 mM glucose. Under the condition of pH 7.8, the polyAAPBA-grafted SiNWs substrate became glucose responsive, which could capture targeted cells in the absence of glucose and release them in presence of 70 mM glucose. The dual-responsive capture and release of CTCs on this polyAAPBA-grafted SiNWs substrate is noninvasive with higher cell viability of 95%. [ 123 ]

Challenges and Future Perspectives
In the previous sections, we described various advanced materials interface mainly based on microfl uidics, nanostructured substrates, and micro-nuclear magnetic resonance systems for CTCs capture and detection. Although promising results have been achieved by these interfaces in terms of capture efficiency and detection sensitivity, most of them still remain in the laboratory level and little of them unequivocally shows clinical validity and utility. [ 124 ] Heterogeneity among CTCs is a problem that cannot be ignored for CTCs isolation. CTCs always express variable biomarkers on their membrane, which affects their morphology and characteristics and makes cell-to-cell variations occur in same cancers, same patient or even within a single blood draw. [125][126][127] Therefore, effi cient CTCs capture and isolation are challenging due to this heterogeneity of CTCs, indicating that not a single cell surface biomarker can confi dently be used for total CTCs isolation. That is why current widely used EpCAM antibodies-based CTCs capture systems do have concerned limitations in clinical applications. Similarly, the immunological capture methods predominantly used in nanostructured substrates and nuclear magnetic resonance-based platforms do have similar concerns. By exploiting the inherent unique biophysical properties of CTCs, label-free microfl uidic strategies seems to have greater potential in clinical capture and isolation of CTCs www.MaterialsViews.com www.advancedscience.com compared to the currently utilized biomarker-based immunological methods by which only a subset of CTCs expressing the selected surface markers are isolated. However, the label-free microfl uidic approach might also introduce false positives results in clinical CTCs capture and isolation by capturing cells that may not directly originate from the primary tumors. [ 128 ] In addition, captured CTCs by some label-free microfl uidic platforms are no longer intact after being subjected to shear forces, thus making subsequent CTCs identifi cation and detection diffi cult.
Beyond the issue of capturing and isolating CTCs, achieving a better understanding of the molecular characteristics of CTCs is also important from which new biomarkers for effi cient CTCs capture and isolation can be discovered. However, traditional CTCs molecular analysis is always performed by using large ensembles on the order of 10 3 -10 6 cells, thereby only giving the average genotypic or phenotypic characteristics of the cell population. In addition, the way the isolated cells are cultured to expand CTC numbers is not recommended, given that cancer cells have a feature that modifi es their characteristics to survive when the surrounding microenvironment is changed. [ 129 ] Single cell analysis has been widely used to explore cellular heterogeneity in gene and protein expressions responsive to environmental change and chemotherapeutic stimuli, providing an effi cient way for CTCs heterogeneity study. [ 130,131 ] Therefore, rationally designed platforms with highly effi cient CTCs capture and single cell evaluation ability are expected to be promising tools for future CTCs study. Learning from the experiences of previous literatures reviewed above, single-cell evaluation technique such as laser scanning cytometry and microfl uidic systems with nanostructure arrays may be a good candidate for the expected objective, given that microfl uidic techniques offer effi cient label-free CTCs separation while the existence of nanostructure arrays confi ne the CTCs migration and enhance the interactions between target CTCs and microfl uidic surface. Furthermore, in order to achieve highly effi cient CTCs capture, multi-biomarkers of CTCs (e.g., EpCAM, HER2, EGFR, and MUC-1) can be patterned to the different regions of microfl uidic system, by which CTCs with different biophysical and biochemical properties can be isolated. Moreover, with the help of single-cell evaluation technique, captured CTCs can be further sensitively detected and characterized by immunological and molecular methods at the single-cell level. (