Facile, rapid and efficient isolation of circulating tumor cells using aptamer-targeted magnetic nanoparticles integrated with a microfluidic device

Facile and sensitive detection and isolation of circulating tumor cells (CTCs) was achieved using the aptamer-targeted magnetic nanoparticles (Apt-MNPs) in conjugation with a microfluidic device. Apt-MNPs were developed by the covalent attachment of anti-MUC1 aptamer to the silica-coated magnetic nanoparticles via the glutaraldehyde linkers. Apt-MNPs displayed high stability and functionality after 6 months of storage at 4 °C. The specific microfluidic device consisting of mixing, sorting and separation modules was fabricated through conventional photo- and soft-lithography by using polydimethylsiloxane. The capture efficiency of Apt-MNPs was first studied in vitro on MCF-7 and MDA-MB-231 cancer cell lines in the bulk and microfluidic platforms. The cell capture yields of more than 91% were obtained at the optimum condition after 60 minutes of exposure to 50 μg mL−1 Apt-MNPs with 10 to 106 cancer cells in different media. CTCs were also isolated efficiently from the blood samples of breast cancer patients and successfully propagated in vitro. The isolated CTCs were further characterized using immunofluorescence staining. The overall results indicated the high potential of the present method for the detection and capture of CTCs.


Introduction
Circulating tumor cells (CTCs), including viable or apoptotic cancer cells released from the primary and metastatic tumor sites into the peripheral blood or lymphatic system, play a critical role in cancer occurrence and metastasis. 1,2 This type of cancer cells has been considered recently as an important indicator or biomarker for the early detection and prognosis of cancers. 3,4 In this regard, the noninvasive isolation of CTCs, a kind of liquid biopsy, has received widespread attention for detection, characterization, and monitoring of cancers. 5,6 However, the rapid and accurate isolation of CTCs is a serious challenge due to their extremely low abundance and heterogeneous nature. Therefore, tremendous efforts have been conducted toward the development of simple, convenient and efficient methods to solve these limitations. 7 To isolate CTCs from the other cells present in patient circulation, the difference between physical and biological properties of the tumor cells compared to the normal blood cells can be targeted. The physical properties which have been utilized for isolation of CTCs are density, size, and surface electrical properties. 8 Generally, the density of CTCs are lower than red blood cells while it is close to mononuclear white blood cells' density. Additionally, most of the circulating tumor cells are larger than blood cells and have higher level of surface foldings, causing a differential surface ion accumulation compared to the blood cells, when they are located in an electric eld. 9 Although all of these physical properties have been extensively used for separation of CTCs from the patients' circulation, 10 in many conditions, mononuclear white blood cells are also separated with CTCs due to their similar physical properties, which makes clinical decision challenging. 2 On the other hand, specic biological properties of CTCs can be targeted using affinity-based approaches. In affinity-based methods, CTCs are captured by the surfaces or particles tagged with specicly designed biomolecules toward the specic surface markers of CTCs. 11 Despite physical separation approaches, high specity of biological methods made them more successful in clinical trials. As a result, the only FDA approved liquid biopsy platform by this time is CellSearch, which utilizes immunomagnetic isolation of CTCs. 12 The immune-magnetic isolation of CTCs is one of the most attractive methods developed until now. The established method of magnetic separation has found widespread clinical applications due to its simplicity, low cost and minimal adverse effects on the captured cells. 13,14 Owing to the use of specic and high-affinity interactions between the recognition agents and cancer cell receptors as well as the efficient magnetic elds, this method is appropriate for the facile, specic and precise detection of CTCs. 15,16 Generally, antibodies with high affinity against specicly expressed receptors on the surface of CTCs are tagged into magnetic micro-or nano-particles and mixed with the body uid to bind into the tumor cells. 17-21 Subsequent application of a magnetic eld can attract the CTC-attached magnetic particles and separate them from the normal cells. However, the clinical application of antibodies has been considerably limited due to their high cost and instability. On the other hand, the multiple batch processing of large volume blood samples with numerus normal blood cells and rare CTC may result in depletion of CTCs and reduced accuracy. Therefore, tremendous efforts have been conducted toward the development of alternative recognition agents and processing methods with comparable sensitivity and specicity. 13 To address the challenge associated with application of antibodies, DNA aptamers have been considered as an attractive alternative for the reliable detection of various targets such as biomolecules, ions and whole cells. [22][23][24] This newly emerged class of nucleic acid ligands, known as the antibody-like molecules, have found growing applications in diagnosis and therapy due to their advantages such as small size, facile synthesis and manipulation, high thermal and chemical stability, low immunogenicity, and high binding affinity and specicity. 25 The increasing reports about the development and use of specic aptamers against different cancer cell receptors such as EGFR, 26 EpCAM, 27,28 PSMA, 29 PTK7, 30,31 sgc8c 32,33 and sLex-AP, 34 clearly indicate high potential of aptamers as the recognition agent.
Mucin 1 (MUC1) is a high molecular weight and glycosylated transmembrane protein belonging to the mucin family that is expressed in the majority of human epithelial cells with a dramatic overexpression in epithelial tumor cells, especially breast adenocarcinoma that has been attributed to their tumor aggressiveness. [35][36][37][38] The circulating MUC1 fragments in the bloodstream of cancer patients are commonly used as a biomarker for determination of cancer stage and monitoring of therapy responses. 39 Among 75 tumor-associated antigens studied by the National Cancer Institute (NCI), MUC1 has been reported as the second-best potential target for the development of vaccines against cancers. 40,41 Furthermore, a short singlestranded DNA aptamer with high affinity for the MUC1 receptor has been reported previously. 42 Due to these unique properties, MUC1 receptor was selected in this study as the target for detection and capture of CTCs based on this anti-MUC1 aptamer.
To harness the full potential of CTCs separation approaches with high accuracy, microuidic devices have been implemented. 10 The continuous ow of body uids inside microchannels reduce the size scale of separation systems down to the dimentions comparable to the cell size, enhancing the possibility of the cells exposure to the separation eld and consequently their isolation in designated microchannels. 43,44 Furthermore, the continuous nature of assays performed using microuidic tools provides the opportunity of their serial integration for multiple cell manipulation steps to increase the accuracy of CTCs isolation and detection without several batch processing steps. 45 However, the serial integration usually comes with a cost of system complexity and higher chance of clogging due to the presence of multiple connections.
In the present study, a novel CTCs capture platform was developed based on CTCs targeting with the MUC1-aptamer conjugated MNPs within a microuidic device. A new and simple approach including the covalent attachment of MUC1 aptamer into the surface of silica coated MNPs through glutaraldehyde linker was employed for development of the stable and functional nanoplatforms. An microuidic network with integrated mixing, sorting and separation modules in a single microchip was also developed and used to investigate the efficiency of the integrated system for CTCs isolation. Considering the overexpression of MUC1 receptors on the surface of breast cancer cells, MCF-7 and MDA-MB-232 cell lines were used as the positive controls. Regarding the expression of MUC1 by the normal epithelial cells and broblast cells, 46 the potential false detection of these cells should be considered. Therefore, the capture efficiency of normal human umbilical vein endothelial cells (HUVEC) and cancer-associated broblast (CAF) cells was also investigated, as the typical epithelial and broblast cells, respectively, with the critical role in the invasion and metastasis of cancer cells. 47,48 In our opinion, this new platform has the potential to develop a simple, fast, precise and cost-effective method for CTCs isolation.
EasySep™ Direct Human CTC Enrichment Kit was purchased from STEMCELL Technologies Inc. (Vancouver, Canada). SU-8 negative photoresist was purchased from Microchem (Sunnyvale, CA, USA). Sylgard 184 was purchased from Dow Corning (Midland, Michigan, USA). All of the aqueous solutions were prepared with double-distilled water.

Synthesis and surface engineering of MNPs
Magnetite nanoparticles were synthesized based on the chemical co-precipitation method according to the previous report. 49 Briey, FeCl 3 and FeCl 2 (molar ratio of 2 : 1) were dissolved in 200 mL distilled water and degassed under nitrogen atmosphere at 60°C for 20 min. Aer the addition of 6 mL ammonium hydroxide (25% w/w), the reaction was proceeded under sonication and stirring at 60°C for 60 min. The resulted nanoparticles were separated magnetically and washed with deionized water to decrease the pH of the solution to 7. For silica coating, the resulted nanoparticles were added into 40 mL absolute ethanol and completely dispersed by sonication under the nitrogen atmosphere. Then, 6 mL deionized water, 3 mL ammonium solution (25% w/w), and 400 mL TEOS were sequentially added into the solution under vigorous stirring and the reaction continued for 5 h at room temperature. The silicacoated MNPs (SiMNPs) were separated, washed with deionized water and dried at 40°C overnight before functionalization with amine groups. For this aim, 20 mg MNPs were rst dispersed in 20 mL ethanol under sonication and 200 mL APTES was added into the solution and reuxed at 60°C, overnight. The resulted amine-functionalized MNPs were separated, washed with deionized water and dispersed in 10 mL phosphate buffer (50 mM, pH 7.0) containing glutaraldehyde (2%, v/v) and incubated at 25°C under stirring for 5 h. MNPs were then separated magnetically, washed with PBS and dispersed in 1 mL phosphate buffer (50 mM, pH 8.5). The NH 2 -terminated anti-MUC1 aptamers were subsequently added into the solution and incubated under gentle mixing at 25°C overnight. The aptamer targeted MNPs (Apt-MNPs) were then separated magnetically, washed and dispersed in PBS (pH 7.4) and stored at 4°C before use. The efficiency of the reaction was determined by measuring the concentration of aptamer in the solution before and aer conjugation using an Epoch micro-volume spectrophotometer system (BioTek, USA).

Characterization of the nanoparticles
The size and shape of MNPs were studied using a eld emission scanning electron microscope (FE-SEM, Zeiss Sigma VP-500) at an accelerating voltage of 15 kV. The elemental composition of MNPs was determined using X-ray energy dispersive spectroscopy (EDS, Oxford Instrument, UK). Fourier transform infrared spectroscopy (FTIR-6300 spectrometer, Jasco, Japan) was used in a wavelength range of 4000 cm −1 to 400 cm −1 to investigate the surface functional groups of MNPs. The magnetic activity of nanoparticles was studied using a vibration sample magnetometer (VSM; Lake Shore Model 7400, Japan) at 30°C. The particle size distribution of the nanoparticles was determined using ZEN 3600 Zetasizer (Malvern, Worcestershire, UK).

Design and fabrication of the microuidic device
The microuidic device was fabricated using the conventional approach of photo-and so-lithography as described previously. 50 Briey, the master mold was fabricated by patterning SU-8 photoresist on a silicon wafer. Subsequently, the microchannels were formed by molding Sylgard 184 reagents, according to the manufacturer's instructions (Dow Corning, USA). A 10 : 1 mixture of base and curing agent was poured on the master, degassed and baked for 30 min at 80°C on a hot plate (RH digital, IKA, Germany). The cured elastomer was then peeled, punched to open the desired inlets and outlets and cleaned with adhesive tapes. The microchannels were then sealed by bonding the polydimethylsiloxane (PDMS) substrate to a glass microscope slide using the plasma activation (BD-20AC, Electro-technic Products, USA). Finally, the bonded devices were baked on the hotplate for 60 min at 80°C to strengthen the bonding and retrieve the hydrophobicity of PDMS surfaces. For CTC capture experiments, the microuidic device was connected to the solution loaded syringes using Tygon® tubing (Cole-Parmer, USA) and metallic connectors, while the injection ow rates were controlled using the syringe pumps (AL-1000, World Precision Instruments, USA).

In vitro studies
The biocompatibility of MNPs was rst studied on the HUVEC and MCF-7 breast cancer cells by MTT assay, according to the previous report. 51 Briey, 10 4 cells were added into each well of a 96-well plate containing 200 mL RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic solution and incubated in a humidied incubator at 37°C under 5% CO 2 . Aer 24 h, different concentrations of MNPs (0, 5, 10, 20, 50, and 100 mg mL −1 ) were added into the individual wells and incubated again for 48 h in the same condition. The medium was then completely removed and 100 mL MTT solution (0.5 mg mL −1 ) was added into each well and incubated again for 4 h at 37°C. Finally, the solution was completely removed and the precipitated formazan crystals were dissolved in 150 mL DMSO and the absorbance was measured at 570 nm. The viability was determined as the absorbance ratio of each treatment and control.
The cell capture efficiency of Apt-MNPs was then evaluated in vitro in the bulk and microuidic platforms. First, the capture efficiency of MCF-7 cells was studied using different incubation periods (10,20,30,60, and 120 min), various concentrations of Apt-MNPs (10, 20, 50, 100 and 200 mg mL −1 ) and two media (PBS buffer or RPMI 1640 medium). The capture efficiency was then determined at the optimum condition using different numbers (10, 50, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 cells in 1 mL PBS) of MCF-7 and MDA-MB-231 cancer cells. The number of captured and noncaptured cells were counted using an optical microscope (Leica, UK). The selectivity of Apt-MNPs was also studied in vitro on HUVEC and CAF cells at the same condition. The cell capture efficiency of Apt-MNPs was also studied in conjugation with a microuidic system by using 50 mg MNPs and 10 4 to 10 6 MCF-7 cancer cells in PBS buffer. The captured MCF-7 cells were then washed with PBS buffer and cultured in RPMI 1640 medium supplemented with 10% FBS and 1% antibiotic solution and incubated in a humidied incubator at 37°C under 5% CO 2 to evaluate their viability and proliferation potential.

CTCs detection and capture from the blood samples of patients
The capture efficiency and specicity of Apt-MNPs were studied using peripheral blood samples of breast cancer patients and healthy donors and compared with EasySep™ Direct Human CTC Enrichment Kit. Aer incubation of 5 mL blood samples with Apt-MNPs (50 mg mL −1 ) for 60 min, the labeled CTCs were magnetically isolated, washed with PBS and enumerated. The immunouorescence (IF) staining was used for CTCs identication according to the previous report 52 with some modications. Briey, the captured cells were rst xed and permeabilized via incubation with 2% paraformaldehyde and 0.1% Triton-X 100, respectively, for 10 min. The cells were then incubated with Hoechst 33 258 solution, FITC-labeled anti-human CD326 (EpCAM) and PE-labeled anti-CD45 antibodies at 37°C for 30 min. The stained cells were then washed with PBS and imaged by an inverted uorescence microscope (Nikon, Eclipse Ti-U) to identify the captured CTCs. Hoechst + / EpCAM + /CD45 − cells were considered as CTCs while Hoechst + /EpCAM − /CD45 + cells were considered as the leukocytes.

Statistical analysis
The in vitro cell studies were carried out in triplicate and the results were presented as the mean value ± standard deviation (SD). The data analysis was performed by Student's t-test (Microso Excel, Microso Corporation, USA) and P values of less than 0.05 were considered as statistically signicant.

Synthesis and characterization of MNPs
In the present study, MNPs were used for CTCs isolation due to their advantages including simple synthesis and functionalization, biocompatibility, superparamagnetic behavior, and also their simple use under the magnetic elds. These unique properties make MNPs an excellent candidate for various biomedical applications such as cell isolation and labeling, targeted drug delivery, imaging, diagnostics and therapeutics. 53 CellSearch®, the only FDA approved kit for CTCs isolation, has also been developed based on the magnetic beads functionalized with a specic antibody against the epithelial cell-adhesion molecules (EpCAM). However, some related challenges with the CellSearch system, especially the limited sensitivity, time-consuming operation, and considerable cost, still need to be solved. 54 Therefore, in the present study, we tried to use the simple and cost-effective methods with the minimum toxicity issues for development of a new approach of CTCs isolation.
The successful application of MNPs mainly depends on the appropriate selection of their physicochemical properties such as size, shape, chemical composition, surface charge and chemistry. 14 As illustrated in Scheme 1, the synthesized MNPs using the co-precipitation method were rst coated with a silica layer to provide the appropriate surface chemistry for further conjugation to the amine-terminated aptamers. The surface chemistry is one of the most important criteria that signicantly affect the biocompatibility, molecular interactions, further functionalization and efficiency of nanoparticles especially in biological uids. 14 Among various polymeric and inorganic materials that are commonly used for surface coating of MNPs, silica is a good candidate due to its biocompatibility, easy modication, less adverse effects on the magnetization and also low costs that make it suitable for commercial applications. 55 The results of the physicochemical characterization of SiMNPs are presented in Fig. 1. FE-SEM image (Fig. 1A) indicates the relatively monodisperse and semi-spherical shape of MNPs with sizes larger than 50 nm. The high surface area of nanoparticles could improve their interaction with the cell surfaces and leads to the efficient capture of cancer cells. 56 The elemental analysis (Fig. 1B) 58 In order to covalently bind the amine-functionalized aptamers to the surface amine groups of MNPs, a highly reactive dialdehyde reagent, glutaraldehyde, was used as the homobifunctional crosslinker. The new and weak band at 1685.4 cm −1 is attributed to the formation of an amide bond between the amine groups on the surface of nanoparticles and the aldehyde group of glutaraldehyde. 59 The subsequent reaction between another terminal aldehyde group of glutaraldehyde and amine group of aptamers led to the covalent attachment of aptamers to the surface of MNPs. This facile and mild Schiff-base reaction using glutaraldehyde linker is commonly used in the biological assays especially for covalent conjugation of antibodies. 60 The concentration of DNA aptamers in the solution before (61 ng mL −1 ) and aer (14 ng mL −1 ) reaction indicated the participation of 77% of total aptamers in the reaction. The calculated efficiency of aptamer conjugation was 2.35 mg aptamer per mg of MNPs.

In vitro one-step capturing of cancer cells
The biocompatibility of MNPs was rst studied in vitro on the HUVEC and MCF-7 cell lines. The survival rates of as much as 90.99 ± 1.16% and 91.38 ± 3.27% were obtained aer 48 h exposure of HUVEC and MCF-7 cells, respectively, with 100 mg mL −1 MNPs (Fig. S1 †) indicating the high biocompatibility of MNPs. Therefore, the nanoparticles are suitable for biological applications especially cell labeling and isolation.
To achieve the best condition for capturing cancer cells, the concentration of Apt-MNPs and incubation time were optimized in the rst step. To this aim, 10 6 MCF-7 cells were rst incubated with different concentrations of Apt-MNPs (10 to 200 mg mL −1 ) and also different incubation times (10 to 120 min) in 1 mL of PBS buffer and then separated magnetically and enumerated under the optical microscope. Based on the results (Fig. S2A †), the highest capture efficiency (95.33 ± 2.52%) was obtained aer 60 min incubation of MCF-7 cancer cells with Apt-MNPs and no signicant increase in the capture efficiency was observed by further increases in the incubation times. The optimum concentration of Apt-MNPs was 50 mg mL −1 , as the capture efficiency of 95 ± 2.65% was obtained aer 60 min incubation of 10 6 MCF-7 cells with 50 mg mL −1 Apt-MNPs. Further increase of Apt-MNPs concentration had no signicant effect on the cell capture efficiency (Fig. S2B †). In the next step, the effect of using PBS buffer and RPMI 1640 medium and also the number of MCF-7 cells was studied on the capture efficiency of Apt-MNPs. Based on the results (Fig. 3A), no signicant difference was observed between PBS buffer and RPMI 1640 medium in terms of the cell capture efficiency. Moreover, Apt-MNPs were able to capture more than 90% of the cancer cells in aboundance as little as 10 cells. The efficient detection and capture of MDA-MB-231 cancer cells were also obtained by using Apt-MNPs, similar to the MCF-7  cells (Fig. 3B). The results were comparable with the previously reported efficiency of ∼90% aer 2 h incubation with 100 mg anti-EpCAM antibody conjugated multifunctional magnetic upconversion nanoparticles. 43 The in vitro cell capture studies on HUVEC and CAF cells were also conducted in the optimum condition to evaluate the selectivity of Apt-MNPs. The capture yields of 53.7 ± 3.52% and 63.4 ± 4.04% were obtained aer 60 min incubation of 50 mg mL −1 Apt-MNPs with 10 6 CAF and HUVEC cells, respectively, that were much lower than the capture yields of MCF-7 (95.33 ± 2.52%) and MDA-MB-231 (94.7 ± 3.52%) cancer cells, in the similar condition. Regarding the expression of MUC1 by the normal epithelial cells and also broblast cells, 46 these results indicate the less capture efficiency of Apt-MNPs for CAF and HUVEC cells in comparison with MCF-7 and MDA-MB-231 cells, due to the lower expression level of MUC1 receptor. The results conrm the direct relationship between capture efficiency of cells and the expression level of receptor. MUC1 receptor is overexpressed on the surface of different cancer cells especially breast cancer cells. 61,62 Anti MUC1 aptamer was selected and used in this study due to its high selectivity and affinity (Kd value of 0.135 nM) for the MUC1 receptor 42 in comparison with the corresponding antibody (Kd values of 3-400 nM). 63 As the positive selection of cancer cells is mainly dependent on the frequency of specic receptors on the cell surface, the capture of normal cells by Apt-MNPs is not expected due to the low expression of MUC1 receptors in these cells.
The microscopic observation of captured cells revealed the formation of a continuous and thick layer of MNPs around the cancer cells (Fig. S3 †). This observation indicated the efficient capture of cancer cells with Apt-MNPs which may be due to the enhanced surface area of MNPs and the high affinity of MUC1 aptamers toward the corresponding receptor. The efficient CTCs capture by formation of a stable patch-like multivalent matrix nano-net via the complexes of antibody-targeted MNPs and graphene oxide sheets have also been reported by Lai et al., 64 recently. Since the conjugation method and the type of linker could signicantly affect the structure and appropriate function of DNA aptamers, this result also conrmed that glutaraldehyde is a suitable linker for the aptamer conjugation to nanoparticles. Based on the results, Apt-MNPs could be an excellent alternative to the conventionally used immunomagnetic microbeads for the efficient capture of cancer cells.
The proliferation ability of magnetically separated MCF-7 cells was also investigated in vitro. Most of the captured cells were viable and successfully attached to the surface of culture plate (Fig. S4A †) and proliferated, however, it took several days for the cells to recover and begin to regrow. The proliferated cells displayed a normal morphology similar to the untreated cells (Fig. S4B †). These results also indicate the biocompatibility of MNPs in agreement with the results of MTT assay.
The high stability was another interesting feature of Apt-MNPs such that their capture efficiency was preserved even aer more than 6 months' storage at 4°C. The capture efficiency of 93.67 ± 2.52% was obtained for MCF-7 cells aer 6 months storage of Apt-MNPs at 4°C. Moreover, the long time storage didn't affect the biocompatibility of Apt-MNPs, as the cell viability of 90.7 ± 2.39% aer exposure of 10 4 MCF-7 cells with 100 mg mL −1 Apt-MNPs for 48 h. Therefore, the simple and fast nature of glutaraldehyde-mediated conjugation along with the high stability of the resulted Apt-MNPs make the present method appropriate for widespread applications. Moreover, the glutaraldehyde-functionalized MNPs could be simply targeted by the aptamer immediately before use to achieve the maximum CTCs capture efficiency. It would be mentioned that the storage of Apt-MNPs at −20°C led to the signicant loss of their function and inhibition of cancer cell capture. The use of Apt-MNPs has also been reported recently by Bayramoglu et al. 65 for the fast and sensitive detection of a pathogenic bacterium, Salmonella enterica, in the milk samples. A sensitive colorimetric method has also been reported recently by Shen et al. 66 for the detection of circulating tumor-related materials by using aptamer-functionalized MNPs and alkaline phosphatase signal amplication.

Microuidic assisted capture of cancer cells
The implementation of microuidic platforms demonstrated promising improvements in accuracy of cell separation and sorting assays. 67 Here, we developed a novel integrated micro-uidic device for aptamer-based magnetic capture of CTCs. The integrated device can be used as a point of care system, without the requirement of previous magnetic labeling step. The platform of the microuidic chip used in the present study is illustrated in Fig. 4. The microuidic network consisted of three main modules including a tesla mixer, an inertial focusing channel, and the main magnetic separation section. Tesla mixer was selected as it works in a wide range of ow rates and is designed based on the previously optimized dimensions 68 to maximize the mixing. Cells and Apt-MNPs were rst introduced into the microuidic chip and mixed homogeneously in the tesla mixer module. As a result, CTCs were specically tagged with MNPs via the MUC1 aptamers. The inertial focusing module then sorts all the cells at the center of the microchannel, enabling separation of magnetically tagged CTCs from the sorted cell stream. An optimized asymmetric curving design 69 based on the applied range of ow rates and average cell diameter was used for inertial focusing. Finaly,the magnetic separation module deviated the trajectory of the magnetically labeled cells and enabled their collection in the bottom outlet while the remained fraction of cells was discarded in the waste upper outlet (Fig. 4).
As one of the specic advantages of the developed micro-uidic device, microchanel network was designed somehow that the device work either as an integrated system or with independent modules. To use all three modules, 1-1 and 1-2 inlets were used for the cell containing solution and MNPs solution, respectively, while other inlets were kept plug. For elimination of the mixer module, when MNPs and cells were previously mixed, only inlet 2 was used and other inlets were kept plug. For the application of only magnetic separation module, when lower ow rates are preferred and therefore inertial sorting module is not effective, inlet 3 was implemented. In this condition, sheath ow entering the microuidic network through inlets 3-1 and 3-2 were applied to conne cells containing stream in the center of the magnetic separation microchannel.
The efficiency of the microuidic chip to magnetically isolate the cancer cells was examined either by using the mixer module or without it (aer initial exposure of MCF-7 cells and MNPs for 60 min). The results were almost similar as up to 90% capture yield was obtained when the cells were isolated directly using the microuidic chip or aer primary incubation with Apt-MNPs for 60 minutes. However, when the mixer module is used, a slow ow rate of less than 20 mL min −1 is necessary for the efficient capture of cancer cells as the higher ow rates lead to the accumulation of MNPs and cancer cells and subsequent blocking of microchannels. Therefore, the use of a mixer module is only suitable for the processing of low volume samples as the long process may result in the degeneration of samples. Aer subjecting to the magnetic eld established by a permanent magnet, the cancer cells and MNPs were separated rapidly from the stream and moved toward the eld (Movie S1 and S2 †). This observation further conrmed the efficient and tight binding of Apt-MNPs to the cancer cells as well as the excellent magnetic activity of MNPs. Similar results were also obtained by Wu et al. 70 regarding the integrated use of magnetic and size-selective separation of CTCs and also by Tang et al. 71 with the integrated use of anti-EpCAM antibody functionalized MNPs and microuidic device. A more than 90% capture efficiency of EpCAM positive tumor cells has also been reported further improves the specicity and sensitivity of CTCs capture allowing take the advantages of DNA aptamers over the conventionally used antibodies. The simple use of glutaraldehyde linker leads to the stable conjugation of aptamers to MNPs with appropriate and prolonged functionality. Moreover, the use of Apt-MNPs makes possible the nondestructive isolation of viable CTCs for the subsequent in vitro culture and molecular analysis. The efficiency of the present method for CTCs capture from the clinical samples is comparable with the examined commercial kit. The overall results indicate the high potential of the present method for clinical applications. Based on the results, the integrated use of microuidic chip with Apt-MNPs is useful to achieve a more automated and efficient method, especially for the analysis of lots of large volume samples. The simplicity and cost-effectiveness of manufacturing are other remarkable characteristics of the present device. In conclusion, the simple, rapid and sensitive nature of the present method makes it a suitable candidate for widespread use in clinical purposes.

Ethical statement
The research related to human use has complied with all the relevant national regulations and institutional policies, follows the tenets of the Helsinki Declaration, and has been approved by the ethical committee of Isfahan University of Medical Sciences (IR.MUI.MED.REC.1395.2.233).

Conflicts of interest
The authors declare no conict of interest.