A chip assisted immunomagnetic separation system for the efficient capture and in situ identification of circulating tumor cells.

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Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x

A Chip Assisted Immunomagnetic Separation System for Efficient Capture and in-situ Identification of Circulating Tumor Cells Man Tang, Cong-Ying Wen, Ling-Ling Wu, Shao-Li Hong, Jiao Hu, Chun-Miao Xu, Dai-Wen Pang, Zhi-Ling Zhang*

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The detection of circulating tumor cells (CTCs), a kind of “liquid biopsy”, represents a potential alternative to noninvasive detection, characterization, and monitoring of carcinoma. Many previous studies have shown that the number of CTCs has significant relationship with the stage of cancer. However, it remains notoriously difficult for CTC enrichment and detection because they are extremely rare in bloodstream. Herein, aided by a microfluidic device, an immunomagnetic separation system was applied to efficient capture and in-situ detection of circulating tumor cells. The magnetic nanospheres (MNs) were modified with anti-epithelial-cell-adhesion-molecule (anti-EpCAM) antibody to fabricate immunomagnetic nanospheres (IMNs). IMNs were then loaded into the magnetic field controllable microfluidic chip to form uniform IMN patterns. The IMN patterns maintained good stability during the whole processes including enrichment, washing and identification. Apart from its simple manufacture process, the obtained microfluidic device was capable of capturing CTCs from bloodstream with efficiency higher than 94%. The captured cells could be directly visualized with an inverted fluorescence microscope in-situ by immunocytochemistry (ICC) identification, which decrease cell loss effectively. Besides that, the CTCs could be recovered completely just by the PBS washing after removing the permanent magnets. It was observed that all of the processes showed negligible influence on the cell viability (viability up to 93%) and the captured cells could be re-cultured for more than 5 passages after released without disassociating IMNs. In addition, the device was applied to the clinical samples and almost all the samples from patients showed positive results, which suggested it could serve as a valuable tool for CTCs enrichment and detection in clinic.

Introduction Circulating tumor cells (CTCs) are the tumor cells disseminated from primary or metastasis sites and then travelled through 1 the bloodstream to other tissues of the body. Many recent studies have demonstrated that CTCs are responsible for the 2, 3 initiation and the in-transit spread of metastasis. Clinical studies have also suggested that the number of CTCs in 4, peripheral blood is proportional to the progression of cancer. 5 Therefore, CTCs are promising to become an important kind 6 of biomarker for metastasis, cancer diagnosis and monitoring. Furthermore, CTC measurement and analysis are regarded as “liquid biopsy” and can be accomplished in peripheral blood instead of some harmful operations, which have provided a 7-9 convenient access to screen disease. Compared with the current gold standard which involves removal of tissues or cells from body and examination by experienced pathologists, CTC measurement and analysis would provide a convenient

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, and Wuhan Institute of Biotechnology, Wuhan University, Wuhan 430072, P. R. China. † Electronic Supplementary Informa)on (ESI) available: See DOI: 10.1039/x0xx00000x

and noninvasive method to diagnosis and monitor cancer. Even though CTCs hold great significance, their detection is proved to be formidable challenge due to the fact that they exist at an extremely low concentration in bloodstream which contains about 109 of haematological cells per millilitre.7 As a consequence, it’s desirable to develop novel platforms to realize rapid, accurate and lossless CTCs isolation and enrichment. The presence of microfluidic techniques have paved the way to overcome the hurdle of the scarcity of CTCs in peripheral blood, and exhibited great potential in CTCs isolation and enrichment.10-12 Most of the microfluidic techniques are based on physical characteristics (size,13-15 density,16 electrical properties17) and/or biological characteristics (antibodyantigen reaction10, 18 and aptamer19, 20). Although physicalbased techniques combined with microfluidic systems have shown unique benefit for label-free and high-throughput isolation, these approaches are generally unable to provide adequate resolution between cell populations with similar size and density. One of most prominent microfluidic device for CTCs isolation depends on the special recognition of cell surface markers by antibody or aptamer. Such techniques take advantage of the different expression levels of certain surface antigens between CTCs and background blood cells to achieve

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high specificity and purity. For instance, in 2007, the pioneer Nagrath and coworker had designed ‘the CTC-chip’ which possessed 78,000 anti-epithelial-cell-adhesion-molecule (antiEpCAM) antibody-functionalized microposts to realize the 10 separation of CTCs from blood. After that, they took use of the structure of herringbones to disrupt the streamlines for maximizing collisions between cells and antibody-modified surfaces to improve the capture efficiency. Both of these devices had shown good performance in accurate identification and measurement of CTCs in blood from patients. Based on these two kinds of structures, many other studies had proposed different kinds of microfluidic devices for 21-25 CTC detection and got good results. Nevertheless, a sophisticated operation to recovery CTCs for downstream studies may limit their usage in highly efficient analysis of CTCs in reality. Magnetic separation is an established method which is widely 18, 25-30 used in both bulk and microchip platforms. Due to its easy manipulation and great convenience in coupling with identification methods, an FDA (the U.S. Food and Drug Administration) approved tool for CTC enrichment and 31 7 32 enumeration for prostate, breast and colorectal cancers is TM available, known as CellSearch . As for microfluidic chip, which can effective decrease reagent dosage, have been reported to separate the cell-magnetic bead complexes and non-magnetized white blood cells based on the accurate regulate and control of the magnetic field distribution around 33-35 the chip. In this way, fast and accurate sorting of cells was accomplished, but a pretreatment of conjugating cells and magnetic bead were needed. On the other hand, CTC-related molecular analyses and functional readouts provide more valuable information about tumor biology during the diagnosis. As a consequence, recovering tumor cells from capture substrate is very necessary. Most of studies took use of enzyme to release the 22, 36 cells , which need some specific enzymes that target cell 37 receptors and/or antibodies and some enzymes like “trypsin” might be very harmful to the viability or the structure of cells. Some other methods including photosensitive-induced 38 39, 40 36, cleavage, electrochemical desorption, thermal stimulus 41, 42 43, 44 or using some nanospheres have the possibility to disturb the cell microenvironment. Compared with them, magnetic separation has the potential to be a very helpful tool for cell release due to the property of easy-manipulation. In our previous work, quick-response magnetic nanospheres (MNs) synthesized by the layer-by-layer (LBL) assembly method for rapid, efficient capture and sensitive detection of 45-47 CTCs was reported. The MNs with five-layers of nano-γFe2O3 assembled on their surface had fast binding kinetics and structural stability. The MNs could also be modified with different kinds of biomolecules such as antibodies, avidin and biotin to target specific protein or cancer cells. While in this method, the wall of tube might adsorb some cells and the washing process in tubes with pipette may also damage cell morphology and viability. Moreover, CTC enrichment and identification were separate, which also had the potential to make the captured cell missing and the identification was

proceed in a small polydimethylsiloxane (PDMS) device with a hole stuck on the surface of a glass which might be mistaken by cell overlap. On the other hands, a new approach to control magnetic field distribution on micrometer scale was designed to generate magnetic bead patterns for microfluidic 48 application. Nickel has lager magnetic permeability compared with the buffer (μr (nickel) ≈200, μr (buffer)≈1). When nickel patterns were magnetized, they will generate a high magnetic field gradient around them, which assist to capture magnetic beads at high flow velocity. With similar theory, this kind of microfluidic chip had already been used in 48 forming cell pattern by cell-magnetic beads complex, or 49 isolating simple sequence repeat marker by magnetic bead . While the forming of cell pattern need to couple cells with commercial beads together first, which may due to cell loss. Additionally, during the process of forming cell pattern, cell may also be missed as a matter of fact some of them may buried in the IMN patterns. For this reason, a method which can be used directly in whole blood should be developed to decrease cell loss. In this work, MNs modified with anti-EpCAM antibody (IMNs) were used to capture tumor cells with the assistant of magnetically controlled microfluidic device. The whole course of loading IMNs to form uniform IMN patterns could be finished in less than 15 minutes and the structure of IMN patterns kept still even after experiencing abundant of haematological cells. Compared with the method of modified antibody on the surface of microposts or herringbones structure, this loading IMNs process was much more convenient and time-saving. Then the IMN patterns were used to capture tumor cells without any pretreatments, after washing by PBS for several minutes the capture efficiency could be up to 94%. As the captured cells were fixed on the IMNs patterns in microfluidic chip, washing process was much simpler than that in tubes, which decreased the possibility of cell loss. When moving the permanent magnets away, almost all the captured cells and IMNs could be released by flushing with PBS. Moreover, the majority of captured cells kept viability, or even could be re-cultured for several passages without disassociating IMNs. In addition, as almost all of the IMNs could be flushed out just by PBS washing, this device could be used repeatedly for more than 6 times. Besides that, the captured tumor cells were fixed on the surface of the IMN patterns with the same plane and could be directly used for immunocytochemistry (ICC) identification and enumeration insitu, thus avoiding cell loss caused by cell recovery or cell overlap. To increase its capacity of processing sample, several microfluidic channels could be arranged in parallel flexibly. Furthermore, positive results were obtained from capturing CTCs in the cancer patient samples while negative results from healthy volunteer blood, which suggested this system would be a promising tool for CTC enrichment and detection in clinic.

Results and discussion Characterization of the MNs and IMNs

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Fig. 1 Characterization of the MNs. (A) Transmission electron microscope (TEM) image of the MNs. The insert is an enlarge image of one IMN. (B) Capture efficiencies of MNs at different attraction times with a commercial magnetic scaffold. (C) Magnetic hysteresis loop of the MNs measured at room temperature. Error bars represent the standard deviations of triplicate experiments.

To get rapid magnetic response, five layers of nano-γ-Fe2O3 were assembled on the surface of the nanospheres through coordination between primary amines of poly (ethylene imine) (PEI) and iron atom of nano-γ-Fe2O3 as reported in our 46 previous work. A layer of silica was then coated outside to fortify its stability and biocompatibility. The TEM image (Fig. 1A) showed that the MNs were uniform in size (356.89 ± 27.53 nm) and well dispersed in water without agglomeration. Almost 100% of the MNs can be captured by a commercial magnetic scaffold (Invitrogen, 12320D, the field strength on the surface of the magnetic scaffold was 325 ± 25 mT) in 1 min (Fig. 1B) showing its rapid magnetic response and uniform magnetism. From the magnetic hysteresis loop (Fig. 1C), it can be seen that the saturation magnetization of MNs was high

(27.66 emu/g) and their retentivity was nearly zero (0.105 emu/g), which verified the MNs’ excellent magnetic property. As we mentioned above, a layer of silica was coated on the surface of the 5-layers-MN to increase its biocompatibility. Additionally, succinic anhydride was introduced to modify MNs with carboxyl. Then MNs were further modified with antiEpCAM antibody, an antibody against to the antigen expressed on the membrane of epithelial cells but not on haematopoietic cells, and used to specifically capture the CTCs from the bloodstream. To confirm the combination of anti-EpCAM antibody and the MNs, Cy3-labeled rabbit anti-mouse IgG antibody was applied to monitor the conjugation. The fluorescence microscope images (Fig. S1, supporting information) proved the MNs were conjugated with antiEpCAM antibody successfully.

Design of magnetically controlled microfluidic chip

Fig. 2 Schematic diagram for the construction of magnetic nanospheres (MNs) based microfluidic device. From top to bottom, the first layer was fluid channel for forming IMN patterns and processing samples. In the middle of the chip, there was ITO glass containing nickel patterns encapsulated in a thin PDMS film. At the bottom of the device, there were two permanent magnets fixed on a glass with opposite NS poles and their gap was 6 mm. Firstly, with the help of permanent magnets, IMNs were loaded in the microfluidic device uniformly. Then sample was added in the reservoir at the other side of the chip and then pulled into the chip. Finally, immunocytochemistry (ICC) identification was carried out directly in the device for CTCs identification and enumeration.

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Fig. 3 Stability of the IMN patterns in the whole blood. (A) Microscopic images of IMN patterns before introducing the whole blood. (B) Microscopic images of IMN patterns when introducing the whole blood. (C) Microscopic images of IMN patterns after introducing the whole blood and washing by PBS.

Three parts composed our device as it mentioned in our previous report.48 As it shown in Fig. 2, from top to bottom, the first layer was fluid channel for forming IMN patterns and processing samples. In the middle of the chip, there was ITO glass containing nickel patterns encapsulated in a thin PDMS film. At the bottom of the device, there were two permanent magnets with opposite NS poles and their gap was 6 mm. The permanent magnets here offered a uniform magnetic field to the fluid channel. The most important part is the second layer which encapsulated nickel patterns in a thin PDMS film. Due to its higher relative magnetic permeability compared to the fluid, nickel patterns were used to increase local magnetic field gradient around them. When the nickel square was magnetized, a high magnetic field gradient was forming around them and the local magnetic field distribution in micrometre scale could be regulated. One of the advantages of the device was that it could form a high magnetic field gradient without the generation of Joule heating which might disrupt the application of the chip for bioanalysis. We designed a nickel square with 50×50 μm, and the distance between two adjacent nickel squares along the Y-axis direction was 50 μm, and 100 μm along the X-axis direction (We defined the direction along the fluid channel as the X-axis and the direction perpendicular to the fluid channel as the Y-axis). As it shown in Figure S2A, the fabricated nickel patterns were nearly square in shape and uniform. 8×12 of nickel squares was considered as one unit of pattern. When loaded into the chip with a flow rate of about 20 μL/min, the MNs could firmly aggregate between two adjacent nickel squares along Y-axis direction (Fig. S2B supporting information). The whole time of loading IMNs to form uniform IMN patterns was only 15 min. The area of each IMN rectangle were about 6155 ± 412 μm2 and the height of IMN rectangle were calculated to 17.1±4.6 μm, which was very stable (Fig. S3 supporting information). The sample was added in the reservoir at the end of the chip as shown in Fig. 2 and then pulled into the chip to prevent cell sedimentation. After washing with PBS, the number of captured cells on the IMN patterns and uncaptured cells were counted to calculate the capture efficiency. Then the MNs could be totally rushed out by PBS if the two permanent magnets were taken away, so

the device could be used repeatedly. Besides that, as CTCs are existed in blood which is very complex in composition and have high viscosity, it’s very necessary to keep IMN patterns stable during the capture process. As shown in Fig. 3, after processing whole blood and then washing with PBS, the structure of IMN patterns had little difference comparing with the original one. Above all, it can be confirmed that the whole blood could be direct applied to the device without any pretreatment. The anti-EpCAM antibody was used to recognize CTCs since EpCAM was plentifully expressed in nearly universal of epithelial cells, but was absent in haematological cells. Thus the anti-EpCAM coated MNs could specifically capture tumor cells originated from epithelial tissue. When tumor cells were introduced into the chip loaded IMN patterns, they could form cell-magnetic complexes with IMNs and fixed on the IMN patterns. The capture efficiencies were calculated as captured cells number divided to the sum of captured and uncaptured cells number. To demonstrate the impact of the arrangement mode of IMN patterns on cell capturing, we first used a single channel with width in 1 mm to compare the capture ability of two kinds of structures. Structure-1 was arranged the nickel squares in parallel while structure-2 was arranged the nickel squares in stagger. The final structure of the IMN patterns took shape according to the arrangement of nickel patterns. As it showed in Fig. 4A, the structure-2 had a higher capture efficiency (96.0%±1.3% (n=3)) to MCF-7 cells, whereas significantly low capture efficiency (43.0% ± 1.2% (n=3)) was observed in structure-1. Fixing the flow rate on 1 μL/min, Fig. S4 (supporting information) showed the cells motion trajectories in structure-1 and structure-2 were also different: cells in structure-1 were moving straight as laminar flow while in structure-2 they were curving. The trajectories of tumor cells moving in the chip revealed that arranged the nickel squares in stagger could enhance cell contact frequency to the IMN patterns. We also found that the size of IMN patterns formed in structure-2 were bigger than in structure-1, which implied that the contact probability between IMNs and tumor cells

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was higher in structure-2. So the structure-2 was better and was chosen in the following experiments. We then investigated the relationship among the length of IMN patterns, the flow rates of sample introducing and capture efficiencies. Microfluidic channel with 1 mm in width was also applied in this experiment. Cell suspensions (10000 cells/mL) containing EpCAM-positive MCF-7 cells dyed with Hoechst 33342 were introduced into the device at flow rates of 0.5, 1, 1.5, 2, and 3 μL/min. Subsequently, substrateimmobilized cells were imaged and counted under an invert fluorescence microscope. The results are shown in Fig. 4B. When flow rate was fixed on 1 μL/min, with the increase of IMN patterns units, the capture efficiency increased until the length reached to 6 units, at which 95.8% of MCF-7 cells were captured. At the same unit numbers of nickel patterns, capture efficiencies decreased sharply as flow rate increased. It also could be summarized from Fig. 4B that if a faster flow rate for sample introducing was needed, more unit numbers of nickel patterns should be applied. The reason why capture efficiency decreased at high flow rate was that, with the increasing of the flow rate, the interaction time between cell and anti-EpCAM antibody on the surface of IMN patterns was reduced, which made it less possible to form “stable” cell adhesion. According to all above and considering the dosage of IMNs, an optimal unit numbers of nickel patterns and flow rate of sample introducing were settled as 8 units and 1.5 μL/min. Besides the high affinity binding force of anti-EpCAM antibody, the irregular surface on the IMN patterns was also a key factor to enhance the capture efficiency. Compared to the flat PDMS micropillars surface, the roughness of the IMN patterns improve the local topographic interactions between antibody on the surface of IMN patterns and nanoscale component on the cell surface. It was worth to notice that, as Fig. S5 (supporting information) showed, the higher of flow rates, the more disperse of the distribution of captured cells on the IMN patterns. This phenomenon was due to the fact that cells need a longer travel length to offset the time loss in the interaction between cell and anti-EpCAM antibody on the surface of IMN patterns at high flow rate. It was another evidence for the reason why increasing unit numbers of nickel patterns could speed up sample introducing.

Fig. 4 (A) Capture efficiencies of the two kinds of structures to MCF-7 cells. (B) The relationship of the unit numbers of nickel patterns and the flow rates of sample introducing on capture efficiency. Error bars represent the standard deviations of triplicate experiments.

Capability of IMN patterns to capture tumor cells The optimal capture conditions were applied to study the general and specificity applicability of the device. Four groups of tests were set to investigate the specificity of our device: (1) IMN patterns to capture MCF-7 cell, (2) MNs patterns without anti-EpCAM antibody modified to capture MCF-7 cell directly, (3) capturing MCF-7 cells without forming IMN patterns and (4) IMN patterns to capture two kinds of human peripheral blood leukaemia cells, Jurkat T cells and HL 60 cells. As shown in Fig. 5A, the binding between IMNs and MCF-7 cells was specific because the IMN patterns could hardly captured any Jurkat T cells or HL 60 cells, and unmodified MNs captured little MCF-7 cells as well. Besides that, two additional EpCAMpositive tumor cells lines (Hep G2 liver cancer cells and Cal 27 human tongue cancer cells) were introduced to be captured by the device with comparable capture efficiencies (Fig. 5B), which was shown this device could be generally applied to other EpCAM-positive tumor cells lines. Then we introduced 4 6 the samples with 10 MCF-7 and 4×10 Jurkat T cells into this device, the result turned out that the concertation of MCF-7 cells increased for more than 50 times. In this way, we can ensure that this method is more competent to remove 47 haematological cells than our previous work.

Fig. 5 (A) Capture efficiencies of IMN patterns to MCF-7 cells, MN patterns to MCF-7 cells, IMN patterns to Jurkat T cells, IMN patterns (anti-EpCAM antibody) to HL 60 cells and MCF-7 cells with no IMN patterns formed in the chip. (B) Capture efficiencies of IMN patterns to MCF-7 cells, Hep G2 cells and Cal 27 cells. (C) Cell captured/released performance of the device, inserts are the structure of IMNs pattern forming and releasing. (D) Capture efficiencies with IMN patterns at different cell concentrations (0-300 cells mL-1) in four different types of samples: PBS (■), DMEM (●), mixture of MCF-7 and Jurkat T cells (▲), whole blood (▼). Error bars represent the standard deviations of triplicate experiments.

As the MNs could be simply rushed out by PBS after moving the two permanent magnets away, we supposed the device

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could be used repeatedly. About 100 MCF-7 cells were introduced into the chip loaded with IMNs for each tests. The results shown in Fig. 5C revealed the chip could be used for more than 6 times. From the insert of Fig. 5C, we also observed that each time the IMN patterns formed firmly and stability in the chip, and could be rushed out completely. The capture efficiencies for each tests kept well, too. We then applied spiked CTC samples to investigate the capability of capture rare population of tumor cells in complex conditions of the device. We had spiked several MCF-7 cells into 1 mL of PBS, DMEM (maintaining 10% fetal bovine serum and 100 IU/mL penicillin-streptomycin), mixed cell suspension and blood from healthy volunteers to prepare spiked CTC samples. The results in the Fig. 5D showed that the device could capture tumor cells in blood successfully. Regression analyses of capture cells number versus total cells number 2 2 were obtained: y= 0.96x (R = 0.999), y= 0.96x (R = 0.995), y= 2 2 0.95x (R = 1.000), and y= 0.94x (R =0.999) in PBS, DMEM, mixed cell suspension and whole blood, respectively. Furthermore, the capture efficiencies in different conditions were analysed and the results showed that there were no significant differences at 0.95 confidence level (P=0.273> 0.05). All of above indicated complex conditions had little effects on the IMN patterns loading in the chip and the device had the ability to capture rare tumor cells in untreated blood directly. It also proved that our device could handle the whole blood directly without any pretreatment processes.

introduced into the device using the above-optimized conditions and reacted with IMN patterns. After washing, a three-color immunocytochemistry (ICC) method was employed to identify and count MCF-7 cells. The three-color immunocytochemistry consists of DAPI (blue, a kind of nuclear staining), fluorescein isothiocyanate (FITC)-labelled antiCytokeratin 19 (CK19, green, a marker for epithelial cells) and allophycocyanin (APC)-labelled anti-CD45 (red, leukocyte common antigen antibody). As shown in Fig. 6, CTCs were defined as DAPI+/CK 19+/CD 45− cells with the size up to 10 μm while white blood cells (WBCs) were DAPI+/CK 19−/CD 45+ cells with the size down to 15 μm. We also observed from the microscope that almost all the captured cells fixed on the IMN patterns in the same plane. In this way, it could make sure that the device could be successful used for in-situ ICC identification without flushing them out of the chip. The process of flushing captured cells out of the chip would decrease the number of captured cells or influence the activity of captured cells. And the process of identification by the 47 PDMS hole as our previous work might be disturbed due to the possibility of cell overlap. Generally speaking, in our method, CTCs can be directly captured in whole blood without any pretreatments and the captured CTCs could be analysed in the chip without releasing, which effectively avoided cell loss and kept cell activity during the whole process. Integration of high-throughput microfluidic device

ICC identification of tumor cells in mimic clinical samples

Fig. 7 (A) Schematic diagrams for combining different

blood samples and identified with the three-color ICC identification. Nucleus (DAPI): excitation 405 nm, emission 447 ± 30 nm band pass. CK 19 (FITC): excitation 488 nm, emission 525 ± 25 nm band pass. CD45 (APC): excitation 605 nm, emission 685 ± 20 nm band pass. Merge: merge of Nucleus (DAPI), CK 19(FITC), and CD45 (APC).

numbers of 2 mm-width-channels chip in parallel. (B) Photograph of the microfluidic device which arranged four 2 mm-width-channels chips in parallel. (C) Relationship between the width of chips (the width of the chip means the sum width of all the microfluidic channels arranged in parallel) and the flow rates of cell introducing. (D) Capture efficiencies in different width of chips with corresponding flow rate. Error bars represent the standard deviations of triplicate experiments.

MCF-7 cells were suspended into healthy volunteer’s whole -1 blood with a concentration of approximately 500 cells mL to mimic clinical samples. The mimic clinical samples were

Due to the limitation of the throughput for sample introducing, it was hard to capture rare CTCs in bulk blood effectivity and rapidly. To solve this problem, we tried to arrange several

Fig. 6 Microscopic images of cells captured from mimic clinical

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fluidic channels in parallel to increase the capacity of the device. As shown in Fig. 7A, one, two, three and four 2 mmwidth-channels arranged in parallel were test in experiments, and each of them had a proportional relationship to the velocity of the inlet with preferable capture efficiencies (Fig. 7B). When there were four 2 mm-width-channels were arranged in parallel, the introducing time of sample of 800 μL could be decreased from 800 min to 100 min. According to that, we could enhance its capacity of sampling by arranging different numbers of microfluidic channels in parallel, so that applying our device to the whole blood in bulk with high efficiency was feasible. Cell viability and re-culture The identification of captured cells viability was measured directly in the chip, Calcein AM and propidium iodide (PI) were used to distinguish live or dead cells. Calcein AM is a kind of dye which can traverse the live cell membrane and hydrolysed by esterase to form calcein with green fluorescence, while PI can’t get through live cell membrane but can enter into dead cells to embed into DNA and then emit with red 10, 50 fluorescence. In this way, we can distinguish the live and dead cells by the colour of fluorescence (Fig. 8A). The viability rate was calculated to 93.1 ± 2.6%, which indicted that most of the MCF-7 cells remained viable after experienced multiplexed capture and washing processes. Since many downstream assays of the CTCs should take outside of the chip, it’s necessary to recovery and re-cultured the captured cells to proliferate for further study. In our experiments, IMNs can help us with easy-manipulating the captured cells. To determine whether captured cells could be re-cultured, 10000 MCF-7 cells were spiked into DMEM and introduced into the chip to be captured by IMN patterns. When we moved away the permanent magnets, the captured cells and IMN patterns could be flushed out by DMEM without any residual and then collected into a 96-well plate for re-culture. As shown in Fig. 8B-E, even the captured cells still linked with IMNs, they could adhere well and proliferate for several passages without any discernible changes in behaviour, which also indicted the harmless of the IMNs to cells.

Fig. 8 Viability of the captured MCF-7 cells. (A) Fluorescence microscopic image of the captured cells in chip stained with Calcein AM (green, live) and PI (red, dead). (B)Microscopic images of the captured tumor cells which were just attached to the flask wall (C), reached confluence after two times of passages (D), reached confluence after three times of passages (E), and reached confluence after five times of passages.

Capture and identification of CTCs from patient peripheral blood samples The blood samples collected from patients with advanced metastatic cancer were analysed for CTC enumeration using the above-optimized conditions. Specifically, a high-through microfluidic chip which containing four 2 mm-width-channels microfluidic channels was used to capture the CTCs from patient peripheral blood samples without any pretreatment process. About 600 ~ 800 μL of whole blood were pumped into the chip loaded IMN patterns in each studies. After washing, a commonly used three-color immunocytochemistry method was utilized in the device to identify and enumerate CTCs from nonspecifically trapped white blood cells (WBCs). CTCs were defined as DAPI+/CK 19+/CD 45− cells with the size up to 10 μm while WBCs were DAPI+/CK 19−/CD 45+ cells with the size down to 15 μm. An inverted fluorescence microscopy was employed to quantify the number of CTCs. As control, we investigated the possibility of capturing CTCs from 3 healthy people’s blood sample. The results were summarized in Table S1 (supporting information). Each blood samples from 10 patients with metastatic were discovered CTCs, and negligible CTCs were detected in the 3 healthy people. In this case, our device was successfully applied to samples in clinic without any pretreatment and could also be used to analysis captured CTCs in-situ, which might be a valuable tool for CTCs enrichment and detection in clinic.

Conclusions In conclusion, we had successfully developed an innovative cell capture, in-situ identification and recovery method. This method integrated the features of magnetically controlled microfluidic chip and IMNs to forming a new kind of IMN patterns with stability, which helped to efficiently capture and in-situ identification of CTCs without any cell loss process. The MNs were fabricated according to a mature LBL assembly method reported by our group and the device was developed to precisely control the magnetic field distribution. Combining these two kinds of technologies, a new type of microposts was made by IMNs. The IMN patterns kept good stability in the whole process of capture and detection CTC in blood, which enable to capture cells effectively even when there were few tumor cells in blood. Moreover, the IMNs had negligible disruption to the viability and functions of cells, and could be directly re-cultured in 96-well plates without disassociating IMNs. The way to combine cell capture and identification into one chip can decrease cell loss efficient and easy to observe under microscope without the influence of cell overlap. Besides that, the device was successfully employed for enriching and detecting CTCs from patient peripheral blood samples, which exhibited its potential application in CTC studies. It is conceivable that if the device was applied to CTCderived molecular analysis, it would provide valuable insight into tumor biology. Above all, this device would be a promising tool for CTC lossless enrichment and detection.

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Cell culture MCF-7 cells, Hep G2 cells and Cal 27 cells were all cultured in common cell culture flasks at 37°C with DMEM supplemented with 10% fetal bovine serum and 100 IU/mL penicillinstreptomycin in a humidified atmosphere with 5% CO2 (in air). The cells were stained with Hochest 33342 for 30 min and then washed five times in 0.01 mol/L phosphate-buffered saline (PBS, pH 7.2). After detached by trypsin solution, the cells were collected by centrifugation at 1500 rpm for 5 min at room temperature. Finally, the cells were translated into 1×PBS which containing 1% Hydroxyl Propyl Methyl Cellulose (HPMC) to prevent cell sedimentation during the injecting process. Jurkat T cells and HL 60 cells were both cultured in common cell culture flasks at 37°C with 1640 supplemented with 10% fetal bovine serum and 100 IU/mL penicillin-streptomycin in a humidified atmosphere with 5% CO2 (in air). Both of them were also stained with Hochest 33342 for 30 min and washing by centrifugation at 1500 rpm for 5 min at room temperature. After that, the cells were translated into 1×PBS which containing 1% Hydroxyl Propyl Methyl Cellulose (HPMC) to prevent cell sedimentation during the injecting process. Magnetic nanospheres fabrication and modification MNs were fabricated by LBL method according to our 46 published work. Making use of emulsifier-free polymerization method, the poly (styrene/acrylamide) copolymer nanospheres (Pst-AAm-COOH) were fabricated firstly. Then PEI (low MW 25 kDa) was coated on the surface of Pst-AAm-COOH nanospheres as a foundation layer to react with nano-γ-Fe2O3 in hexanol. The second layer was formed similarly: another kind of PEI (high MW 750 kDa) kept on coating on the surface of the nanospheres in PBS and then attach to an additional layer of nano-γ-Fe2O3 in hexanol. Repeat this step for four times, and then five-layer magnetic nanosphere was assembled successfully. After that, an outer shell of silica was coated on the surface of the magnetic 51 nanoparticle by a seeded-growth method. After coated with a silica shell outside of the nanosphere, amino-terminated silica-coated nanospheres were purified with centrifuge and then dispersed into N, Ndimethylformamide (DMF) containing succinic anhydride to form carboxyl group for further reactions. Carbodiimide chemistry was used to cross-link amines of the antibody with the carboxylic acid groups on the surface of MNs-COOH. The resultant immunomagnetic nanospheres (MNs-anti-EpCAM antibody, IMNs) were dispersed into PBS for reserve. Microfluidic device design and fabrication The fabrication procedure of the nickel patterns on the bottom 48 of the microchannel was shown in our previous work. And the microfluidic chip was fabricated by the standard soft lithography method. First, the AZ-50XT photoresist was prepared on a clean, smooth silicon wafer by spin coating

method. And with the help of UV exposure, a 40-μm-thick, 1mm-width master was obtained. PDMS prepolymer (10:1 w/w of the RTV615A/RTV615B) was poured onto the silicon master and baked at 75°C for 4 h. Then the solid polymer was peeled off the photoresist structures and punched with a blunt needle for inlets and outlets. The final chip was assembled in three layers. Two permanent magnets were fixed parallel under a clean glass slide with double-sided tape. The nickel pattern was encapsulated in a thin PDMS film above the ITO glass. A PDMS fluid layer irreversibly bonded with the second nickel pattern layer. With the help of permanent magnets, a certain amount of IMNs were loaded into the chip to generate uniform IMN patterns with a flow rate of 20 μL/min. Capture of tumor cells with IMN patterns Four groups of MCF-7 cell-spiked samples were prepared as follows: several Hochest 33342-stained MCF-7 cells were respectively spiked into 1×PBS, DMEM (maintaining 10% fetal bovine serum and 100 IU/mL penicillin-streptomycin), HL 60 cell suspension, and whole blood. Then the samples were introduced into the device to be captured by IMN patterns. After the cells were captured, the microfluidic channel was washed by PBS. Captured and uncaptured MCF-7 cells were all counted to calculate the capture efficiencies. To investigate the effect of flow velocity and the length of nickel patterns on the capture efficiency, MCF-7 cells were introduced into chip of different numbers of nickel patterns units with different flow rates. Capture efficiencies of two other kinds of tumor cells (Cal 27 cells and Hep G2 cells) were also calculated to test the general applicability of this method. Two kinds of nonEpCAM expressed cells (Jurkat T cells and HL 60 cells) were used to describe the specificity of this method. Confining liquid which containing 1% BSA and 0.1% Tween-20 was applied to inhibit the blood cells and IMNs attached on substrate of capturing site or the wall of microfluidic channel. Cell capture/release performance in multiple cycles of studies with repeated producing IMN patterns was studied. Whole blood samples were collected from healthy volunteers into EDTAcoated vacutainer tubes, and were used within 24 h. ICC identification of tumor cells in mimic clinical samples MCF-7 cells were suspended into healthy volunteer’s whole -1 blood with a concentration of approximately 500 cells mL to prepare mimic clinical samples. Then IMN patterns were formed uniformly and the mimic sample was introduced with a flow rate of 1 μL/min. After washed by 1×PBS for 5 min with the flow rate of 1 μL/min, the captured cells were fixed with 4% paraformaldehyde (10 min, 20 μL/h), permeabilized with 0.1% Triton-X 100 (10 min, 20 μL/h) and stained with ICC identification consisting of 30 μg/mL DAPI, FITC-labeled antiCK19 monoclonal antibodies, and APC-labeled anti-CD45 monoclonal antibodies (30 min, 20 μL/h). After washing steps, the number of captured cells was recorded and the cell morphology was imaged by an inverted ﬂuorescence microscope. Cells that had round to oval morphology, and positive for DAPI/CK19 and negative for CD45, were identified

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as tumor cells, those positive for DAPI/CD45 and negative for CK19 were identified as white cells.


Cell release and cell viability analysis LIVE/DEAD viability kit was used directly in the chip to show the influence on cell viability of this method. Briefly, the captured cells in the chip were stained with 2 μM calcein AM and 4.5 μM propidium iodide (PI) at room temperature (30 min, 20 μL/h). Then the cells were observed under a fluorescence microscope excited by blue light, and all the captured cells were counted to calculate the viability rate. Furthermore, whether the isolated cancer cells could be cultured and expanded in vitro was investigated. First of all, we need to release the captured cells from the chip. The IMN patterns could be released easily by removing the permanent magnets and flushing out with higher flow rate. In this experiment, captured MCF-7 cells were rushed out by DMEM (maintaining 10% fetal bovine serum and 100 IU/mL penicillinstreptomycin) after removing the permanent magnets, and recovered to be cultured at 37 °C in a 96-well plate. Sterile conditions should be kept during the whole procedure. Detection of CTCs in cancer patient peripheral blood samples Blood samples from 10 cancer patients and 3 healthy volunteers were collected and introduced into the magnetically controlled microfluidic device with uniform IMN patterns without any pretreatment. The blood samples were collected in an EDTA coated vacutainer tube. After washing, fixing, permeabilizing, blocking and staining, the captured cells were observed under a fluorescence microscope. Only the cells with CK19 and DAPI positive but CD45 negative, and their size up to 10 μm were enumerated as CTCs.

Acknowledgements This work was supported by the 863 Program (2013AA032204), the National Natural Science Foundation of China (21475099 and 21175100), and the Natural Science Foundation of Hubei Province (2014CFA003). The blood samples from healthy volunteers were kindly provided by Hospital of Wuhan University and blood samples from patients with advanced metastatic cancer were provided by Hubei Cancer Hospital.

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Highly efficient capture and harvest of circulating tumor cells on a microfluidic chip integrated with herringbone and micropost arrays.

Efficient capturing of circulating tumor cells using a magnetic capture column and a size-selective filter.

Quick-response magnetic nanospheres for rapid, efficient capture and sensitive detection of circulating tumor cells.

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Efficient capture and simple quantification of circulating tumor cells using quantum dots and magnetic beads.

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Acoustic separation of circulating tumor cells.

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Circulating tumor cells capture disease evolution in advanced prostate cancer.

An immunomagnetic separation-real-time PCR system for the detection of Alicyclobacillus acidoterrestris in fruit products.

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Model experiments for immunomagnetic elimination of leukemic cells from human bone marrow. Presentation of a novel magnetic separation system.

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Ex vivo expansion of circulating lung tumor cells based on one-step microfluidics-based immunomagnetic isolation.