Label-free isolation of circulating tumor cells using negative lateral dielectrophoresis- assisted inertial microfluidics

Label-free isolation of circulating tumor cells using negative lateral dielectrophoresis- assisted inertial microfluidics | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Label-free isolation of circulating tumor cells using negative lateral dielectrophoresis- assisted inertial microfluidics Aliasghar Mohammadi, Morteza Safari, Mehdi Rahmanian This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2752830/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Jul, 2023 Read the published version in Microfluidics and Nanofluidics → Version 1 posted 7 You are reading this latest preprint version Abstract In this study, a dielectrophoresis (DEP)-assisted inertial microfluidics methodology was introduced for the the isolation of circulating tumor cells (CTCs) from diluted blood samples. The methodology was based on the negative DEP, provided with the laterally allocated microelectrodes as well as the proper selection of the applied electric field frequency and voltage, to suppress the limited purity arising from the overlapped sizes of CTCs and white blood cells. Initially, the dynamics of 5 and 15 µm polystyrene microparticles within the DEP-assisted inertial microfluidic device were numerically and expimentally investigated. While the dynamics of the larger microparticles was governed by the inertial and DEP forces, those of the smaller microparticles were subject to the Dean drag force. In the absence of the DEP force, the larger microparticles migrate to two stable equilibrium positions corresponding to the upper and lower walls for the microchannel cross-section. In the presence of the DEP force, the equilibrium position corresponding to the lower wall is considerably displaced, while the equilibrium position corresponding to the top wall remains almost intact. Finally, it was found that the methodology outperformed the corresponding solely-inertial methodology in terms of purity for the isolation of CTCs from diluted blood samples. For instance, the purity of isolated MDA-MB-231 spiked in diluted blood samples, at a hematocrit of 1%, by the solely-inertial microfluidic device was 85.3%, while viable CTCs were captured using the DEP-assisted inertial microfluidic device with 94.1% purity at the total flow rate and applied voltage of, respectively, 650 µL min − 1 and 50 V. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Introduction Despite the increasing clinical advances in cancer diagnosis and treatment, cancer has remained one of the deadliest diseases in the world 1 – 2 . The high mortality rate of cancer arises from the aggressive spread of this disease throughout the body through a phenomenon known as metastasis 3 . Metastasis is responsible for more than 90% of cancer mortality. As a matter of course, metastasis is the most important barrier in our combat against cancer; such that cancer mortality does not decrease unless using a reliable biomarker to diagnose metastasis at the early stages 4 – 5 . In the process of metastasis, circulating tumor cells (CTCs) are shed from the primary tumor and conveyed to distant organs via circulation 4 – 5 . Therefore, CTCs play a pivotal role in the development of secondary tumors 4 , 6 . The vitality of metastasis is more clarified when we consider that a 1-g tumor releases more than two million CTCs per day 7 . However, CTCs have a limited half-life and many of them fail to survive the physical tensions in the bloodstream 8 . Nevertheless, few detached survived CTCs can spread cancer by generating tumors in other organs 9 . Apart from the formation of secondary tumors, the lack of a unique prescription for all patients due to the heterogeneity of tumor cells is another challenge, which increases cancer mortality. In this regard, personalized therapy can lead to a shorter and more effective treatment period 10 . The current approaches for cancer diagnosis hinge upon invasive procedures such as tissue and bone biopsy. In this context, enumeration and isolation of CTCs can be an alternative strategy for early cancer diagnosis in a less invasive manner. Moreover, this approach provides a real-time assessment of a patient condition as it simply demands a blood sample or other bodily fluids 11 . In other words, the number of CTCs in the blood could be an indicator of the tumor response to a specific therapy 12 . Furthermore, capturing viable CTCs provides the opportunity to conduct in vitro studies for the development of anti-cancer drugs 13 – 14 . However, the isolation of CTCs is challenging due to their low concentration and size, which overlaps those of white blood cells (WBCs) 15 . Conventional techniques for the isolation of CTCs include density-gradient centrifugation, flow cytometry, and fluorescence-activated as well as magnetic-activated cell sorting. Even though such techniques are well established, they require bulky and expensive equipment as well as detailed protocols. On the other hand, microfluidic-based techniques provide the opportunity to regulate and process a small amount of sample within a short time at a lower cell separation cost. As such, microfluidics has increasingly come into focus for cell separation and manipulation 16 – 18 . In general, microfluidic-based cell separation techniques include two groups of passive and active. Passive techniques rely on differences among some intrinsic properties of cells, such as size, shape, and deformability 19 – 20 . On the other hand, active techniques use external forces for separating cells upon differences in dielectric properties, magnetic susceptibility, compressibility, or refractive index 21 – 25 . Among the passive techniques, inertial-based separations have been in focus for the separation of cancer cells in the last two decades (see section S1.1 of the supporting information for a brief introduction to the inertial migration in microchannels) 26 – 29 . In this context, various microchannels with diverse geometries were reported for the improved isolation of CTCs 12 , 27 , 30 – 36 . In spite of this fact, the contamination of isolated CTCs with WBCs is inevitable in size-based inertial microfluidics separations, due to the overlapped sizes of CTCs and WBCs 37 (see section S1.2 of the supporting information for a brief introduction to WBCs). Moreover, the separation of cells is not easily regulated in inertial-based methods once the microfluidic device geometry is fixed. Keeping this in mind, active methods can be utilized for separating same-sized cells, where the separation performance can be modulated through tuning externally controllable parameters. Nevertheless, active methods are a proper choice for cell separation at limited throughput 38 . Thus, hybrid platforms have been developed for cell separation. A combination of the high precision of active methods and the high throughput of inertial-based methods can result in a powerful cell separation platform. Among the previously reported hybrid systems 39 – 42 , synergizing inertial-based methods with dielectrophoresis (DEP) appears a proper combination (see section S1.3 of the supporting information for a brief introduction to the dielectrophoresis). In this venue, of the studies reported as the inertial-DEP combination was a hybrid inertial-DEP platform 43 , where the DEP was introduced through full coupling with a serpentine microchannel. A sheath flow was used to eliminate the upper inertial equilibrium positions. Then, the DEP force was competing with the inertial lift force along the vertical direction, levitating particles in the vertical direction. Subsequently, differential focusing positions of particles along the lateral direction were attained. The inertial-DEP platform was, however, demanding a careful selection of operational parameters to modulate the equilibrium positions. For instance, the positions were laterally differentiated at specific voltages; otherwise, no lateral differential focusing was observed. As such, there is still a lack of an inertial-DEP platform to easily control the equilibrium positions along the lateral direction. In this study, a spiral microchannel was coupled with laterally allocated microelectrodes to form a, so-called, spiral-DEP platform (Fig. 1 ). The developed platform had many advantages over the current microfluidics particle manipulation methods. This method was based on negative dielectrophoresis such that particles were not exposed to the strong electric fields near the microelectrodes. This is especially important for bioparticles, where prolonged exposure to strong electric fields leads to cell damage. In this context, the developed platform was used to isolate CTCs from blood. The designed microelectrodes were responsible for the deflection of the non-target cells towards the outer wall based on a negative DEP force as well as the crossover frequencies of different bioparticles. All types of non-target cells were removed, irrespective of their size-overlap with target cells, by taking advantage of the selective inertial focusing for the inertial section and negative DEP for the dielectrophoresis section. Thus, WBC contamination was avoided in a label-free approach. The remainder of this study is laid out as follows. Section 2 outlines the design and simulation of the microfluidic device. Fabrication procedures and experiments are discussed in section 3 . Results are provided and discussed in section 4 . Finally, a summary is given in section 5 . 2. Design And Simulation Of The Microfluidic Device In this study, a spiral microchannel with three turns as well as two inlets and two outlets was employed. The inner radius and distance between the turns of the spiral microchannel were, respectively, 6 and 1 mm. Moreover, the width and depth of the microchannel were 500 and 170 µm, respectively. The inner and outer inlets were employed for the injection of the sample and sheath fluid, respectively (Fig. 1 ). The sample was diluted CTC-spiked blood while the sheath fluid was phosphate-buffered saline (PBS). In addition, CTCs and blood cells were to come out of the microchannel through the inner and outer outlets, respectively. In other words, the CTCs and the blood cells are the target and non-target cells, respectively. Although many of the non-target cells are to remove in the spiral microchannel using the Dean-cycle phenomenon (see section S1.1 of the supporting information), such microfluidic devices fail to remove non-target cells whose size overlaps the size of the target cells. In the developed spiral microchannel, CTCs (target) and large WBCs (non-target) focus close to the inner wall, reducing the purity of the isolated CTCs. To increase the purity of the isolated CTCs, the DEP force was employed to push WBCs away from the inner wall. In this venue, the conventional practice to generate such negative DEP force involves embedding microelectrodes on one side of the microchannel. However, this approach was challenging due to geometrical restrictions. As such, an unprecedented microelectrode pattern (figure S1 ) was designed to generate the negative DEP force to repel non-target cells from the inner wall. As depicted in Fig. 2 , the Dean drag and lift forces cancel out each other at the equilibrium positions, such that the microelectrode-generated negative DEP force pushes the cells away from the inner wall. The proper selection of operational parameters solely through experimental tests is time-consuming and costly due to the complex nature of cell dynamics resulting from the inertial lift, Dean drag, and DEP forces. Accordingly, numerical simulations were included to assist in the design of the microfluidic device. In this regard, the details of the simulation procedure are provided in section S2 of the supporting information. Briefly, a three-dimensional direct numerical simulation was conducted to examine the effect of inertial lift, Dean drag, and DEP forces on the micro- and bioparticle dynamics in the spiral-DEP platform. For this purpose, the COMSOL Multiphysics software version 5.6 was used to build a model for solving fluid flow and electric field. Then, the simulated fluid flow and electric field were used to calculate the hydrodynamic (Dean drag, added mass, and lift forces) and DEP forces for the particle tracing analysis of the micro- and bioparticles. 3. Microfluidic Device Fabrication And Operating Procedures The proposed microfluidic device was comprising two parts: the microchannel and the microelectrodes. While the former was fabricated by standard soft-lithography procedure, the microelectrodes were prepared by conventional photolithography. The fabrication procedures are described in section S8 of the supporting information. Two types of experiments were performed with the spiral-DEP platform. In the earlier experiments, the physics of solely-inertial as well as inertial-DEP microfluidics was examined by separating polystyrene microparticles. To this end, a binary mixture of 5 and 15 µm polystyrene microparticles was suspended in deionized water at a volume fraction of 0.1 vol.%. In the latter experiments, the isolation of CTCs was addressed using the spiral-DEP platform with diluted blood samples spiked with the MDA-MB-231 cell line such that the resulting concentration of the CTCs within the diluted blood samples was 10 4 CTCs mL − 1 . The details of the procedures used for the preparation of the non-biological and biological samples are described in section S9 of the supporting information. Also, prepared samples were injected into the microfluidic device based on the operating procedure described in section S10 of the supporting information. It must be noted that throughout all the experiments, the sample flow rate was fixed at 100 µL min − 1 and the sheath fluid flow rate ranged from 150 to 750 µL min − 1 . Meanwhile, three measures were used for the interpretation of the resulting data and assessing the microfluidic device performance: $$\text{E}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}\text{y}=\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{t}\text{a}\text{r}\text{g}\text{e}\text{t}\text{e}\text{d} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}/\text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{n}\text{e}\text{r} \text{o}\text{u}\text{t}\text{l}\text{e}\text{t}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{t}\text{a}\text{r}\text{g}\text{e}\text{t}\text{e}\text{d} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}/\text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{l}\text{e}\text{t}}, \left(1\right)$$ $$\text{P}\text{u}\text{r}\text{i}\text{t}\text{y}=\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{t}\text{a}\text{r}\text{g}\text{e}\text{t}\text{e}\text{d} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}/\text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{n}\text{e}\text{r} \text{o}\text{u}\text{t}\text{l}\text{e}\text{t}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}/\text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{n}\text{e}\text{r} \text{o}\text{u}\text{t}\text{l}\text{e}\text{t}}, \left(2\right)$$ and $$\text{R}\text{e}\text{j}\text{e}\text{c}\text{t}\text{i}\text{o}\text{n} \text{r}\text{a}\text{t}\text{i}\text{o}=1-\frac{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{b}\text{l}\text{o}\text{o}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{n}\text{e}\text{r} \text{o}\text{u}\text{t}\text{l}\text{e}\text{t}}{\text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{b}\text{l}\text{o}\text{o}\text{d} \text{c}\text{e}\text{l}\text{l}\text{s} \text{i}\text{n} \text{t}\text{h}\text{e} \text{i}\text{n}\text{p}\text{u}\text{t} \text{b}\text{l}\text{o}\text{o}\text{d} \text{s}\text{a}\text{m}\text{p}\text{l}\text{e}}. \left(3\right)$$ Moreover, a trypan blue exclusion assay was performed in solely-inertial and inertial-DEP platforms to measure the viability of the CTCs 14 . 4. Results And Discussion The aim of this study is to improve the purity of the CTCs isolation from blood utilizing the synergistic effect of the inertial and dielectrophoresis phenomena. In this regard, the dynamics of different-sized polystyrene microparticles was first simulated to determine their lateral positions at the outlet of the spiral microfluidic device. In the second part of this section, the dielectrophoretic force was applied to manipulate the lateral positions of the microparticles at the outlet. Finally, in the last part of this section, the isolation of the CTCs from blood was experimentally investigated to examine the robustness of the spiral-DEP microfluidic device. 4.1. Dynamics of microparticles in the spiral microfluidic device The dynamics of polystyrene microparticles, with the sizes of 5 and 15 µm, in the spiral microfluidic device was simulated at various flow rates. Figure 3 depicts the distance of the microparticles from the inner wall at the outlet of the spiral microfluidic device. It is observed that the total flow rate considerably modulates the distance of all the particles. This observation is explained by considering the forces governing the dynamics of the microparticles. At the entrance region of the spiral microfluidic device, the particles were focused close to the outer wall with the aid of a sheath flow. In such a situation, the particle motion is governed by the competition between the inertial lift force \({\mathbf{F}}_{\text{l}\text{i}\text{f}\text{t}}\) and the drag force exerted by the Dean vortices \({\mathbf{F}}_{\text{D}\text{e}\text{a}\text{n}}\) . Due to the different scaling of these forces \(\left|{\mathbf{F}}_{\text{l}\text{i}\text{f}\text{t}}\right| \tilde{ \text{d}}_{\text{p}}^{4}\) and \(\left|{\mathbf{F}}_{\text{D}\text{e}\text{a}\text{n}}\right| \tilde{\text{d}}_{\text{p}}\) , the microparticles undergo two different trajectories based on their sizes. For the larger particles, both the forces are relevant. At the central region close to the inner wall, the force field resulting from their superposition brings the larger particles to two equilibrium positions. In spite of this fact, all the larger particles should have time to reach the equilibrium positions. As such, there is a lower limit for the total flow rate to inertially focus the larger particles. This fact is apparent in the focusing of the larger particles, shown in Fig. 3 b. The fosuing position of the larger particles changes with increasing the total flow rate. The dynamics of the smaller particles is, however, modulated by the Dean drag forces rather than the lift forces. In this extreme, the smaller particles follow two symmetrical vortices, remaining randomly distributed. Since the fluid circulates laterally due to the vortices, the smaller particles are in a continuous lateral circulation. Thus, the positions of the smaller particles at the outlet rely on the geometry of the microchannel and the flow rate. For a spiral microchannel with specified geometry, the positions are solely subject to the flow rate. The positions of the smaller particles oscillates as a function of the flow rate (see Fig. 3 ). This fact modulates the separation purity for the larger particles. In order to afford the highest separation purity, the total flow rate must be at specific values to ensure an integer number of, so-called, Dean cycle (see Fig. 3 ). Considering the aforementioned two constarints for the proper flow rate, we opted the total flow rate of 650 µL min − 1 to provide both complete inertial migration and 1 Dean cycle. It must be noted that the total flow rate of 1300 µL min − 1 satisfies the aforementioned constraints, but we selected 650 µL min − 1 as the opted total flow rate. As PDMS deforms with increasing the flow rate, leading to unreliable results owing to changing the dimensions and shape of the microchannels cross-section 44 . After selecting the proper flow rates, the sepration of the 5 and 15 µm polystyrene microparticles using the spiral microfluidic device was simulated, and compared with the experimental results. The separation results from the simulation and experiments are shown in Figs. 4 a- 4 c. It was experimentally obtained that the polystyrene microparticles were separated with the efficiency and purity of 78% and 96.1%, respectively. This finding indicates that the opted flow rates were proper such that the larger particles migrate inertially towards the inner wall while the smaller particles circulate laterally towards the outer wall. Furthermore, our computational results not only confirm this phenomenon but also demonstrate that inertial migration occurs in two stages (see Fig. 4 d), where the larger particles migrate first to an equilibrium perimeter and slowly to the center of the faces 42 , 45 . 4.2. Dynamics of microparticles in the spiral-DEP microfluidic device Figure 5 demonstrates the contributing forces on the dynamics of the 15 µm polystyrene microparticles in the spiral-DEP platform. The equilibrium positions of the microparticles are calculated using the constraint \({\mathbf{F}}_{\text{t}\text{o}\text{t}\text{a}\text{l}}=0\) . It is observed that the equilibrium positions of the 15 µm polystyrene microparticles are modulated in the presence of the DEP force. The real part of the Clausius-Mossotti factor determines the direction of the DEP force. In this context, the real part of the Clausius-Mossotti factor is negative for the 15 µm particles at the frequency of 1 MHz, and the DEP force is directed away from the microelectrodes. In addition, the equilibrium position corresponding to the lower wall is considerably displaced, while the equilibrium position corresponding to the top wall remains almost intact by the DEP force generated from the microelectrodes. The reason is the short-range nature of the DEP force, which exponentially decays by the distance from the microelectrodes. We simulated the dynamics of the 15 µm polystyrene microparticles in the presence of the DEP force. In the spiral-DEP platform, there is no allocated microelectrodes within the initial 1 cm of the spiral microchannel; as such, the lateral position of the microparticles does not change by increasing the voltage within the initial 1 cm. Also, the curvature is less in the initial first turn of the spiral microchannel; thus, the effect of the Dean drag force is significant and the position of the microparticles does not change with varying the voltage in the first 3 cm of the spiral microchannel (region #1 in Fig. 6 ). In the subsequent region, the 15 µm polystyrene microparticles are gradually directed away from the microelectrodes in the presence of the DEP force (region #2 in Fig. 6 ). It is observed that a negative DEP force directs away particles from inertial equilibrium positions in the vicinity of the microelectrodes. In this venue, the effect of the DEP force was experimentally examined on the dynamics of the 15 µm polystyrene microparticles. To this end, a sample of the 15 µm polystyrene microparticles was injected into the spiral-DEP microfluidic device at a total flow rate of 650 µL min − 1 . Meanwhile, the frequency of the applied electric field was set 1 MHz and the voltage increased from 0 to 50 V. Figure 7 depicts the size distribution of the microparticles at the inlet and outlets of the spiral-DEP microfluidic device. The majority of the microparticles (⁓96%) migrated towards the inner wall in the absence of the DEP force, whereas the microparticles start to migrate towards the outer wall with increasing the voltage as a result of the negative DEP force generated by the microelectrodes (Figs. 7 b and 7 c). This indicates that the microelectrodes enable repelling non-target micro- and bioparticles. However, given movie S1 and Figs. 7 b and 7 c, the microparticles did not completely leave through the outer wall as the equilibrium position corresponding to the top wall remains almost intact by the DEP force generated from the microelectrodes (see figure S7). Furthermore, the effect of the DEP force on the dynamics of the 5 µm polystyrene microparticles was computationally and experimentally investigated (see Fig. 8 ). It is observed that the DEP force does not disrupt the Dean cycle and the 5 µm particles migrate to the outer wall (1 Dean cycle) at the total flow rate of 650 µL min − 1 . To understand the physics underlying this observation, recall the scaling of \(\left|{\mathbf{F}}_{\text{D}\text{E}\text{P}}/{\mathbf{F}}_{\text{D}\text{e}\text{a}\text{n}}\right| \tilde{ \text{d}}_{\text{p}}^{3}\) (see equations S4 and S5). Accordingly, the dynamics of the 5 µm particles is modulated by the Dean drag force rather than the DEP force. This indicates that the 5 µm particles are driven by the Dean vortices. We, however, note that the position of the 5 µm microparticles slightly changes with increasing the voltage (see Fig. 8 a). The position of the 5 µm polystyrene microparticles does not notably change until the second turn (region #1), but they, in the third turn, start to migrate towards the outer wall with increasing the voltage (region #2). This fact is associated with the gradual increase of the curvature radius in the spiral microchannel, where the magnitude of the Dean drag force in the third turn is not as strong as that in the first and second turns. Thus, the 5 µm microparticles were slightly pushed towards the outer wall owing to the negative DEP force generated by the microelectrodes. This phenomenon results in the decrement of the width of the microparticle stream at the outlet, which improves the separation purity (see Figs. 8 c and 8 d). 4.3. Spiral-DEP platform in the context of CTCs isolation Figure 9 a shows the measured rejection ratio for the diluted blood samples at two distinct hematocrit levels of 1 and 10% as a function of the total flow rate. The highest rejection ratio was recorded at the total flow rate of ~ 650 µL min − 1 for the considered hematocrit levels. As also revealed by the plots in the figure, the rejection ratio decreases with increasing the hematocrit level. Needless to say, separating the blood cells from the serum is more difficult at higher hematocrits. In the next step, a diluted blood sample at the hematocrit level of 1% was spiked with the MDA-MB-231 cell line and injected into the spiral-DEP platform to separate the CTCs from the blood sample. The influence of the voltage on the separation purity is presented in Fig. 9 d. The purity shows an ascending trend by increasing the voltage. The impact of the DEP becomes imperative with increasing the voltage. This observation is associated with the forces contributing to the cell dynamics in the DEP-spiral platform, which are the inertial lift, Dean drag, and DEP forces. At zero voltage, the CTCs were isolated under the influence of hydrodynamic forces that distinguish cells based on their size. In this case, the purity of the MDA-MB-231 cell line was 85.3%. With increasing the voltage, the DEP force becomes imperative for the bioparticle migration. Therefore, the purity increased to 94.1%. This is mainly because the negative DEP force generated by the microelectrodes contributes to removing larger WBCs that the spiral microfluidic device could not remove. Let us continue with the task of addressing the impact of the DEP force on the separation efficiency of the CTCs. While the efficiency was found 88.0% in the absence of DEP, the efficiency was 89.0% for the total flow rate of 650 µL min − 1 and the applied voltage of 50 V. Therefore, the DEP does not have a considerable effect on efficiency. This fact arises from the selected frequency at which the CTCs experience a relatively small DEP force. Finally, the effect of the cell separation by the spiral-DEP platform on the viability of the isolated CTCs was explored. During the operation, the cells experience stresses from two distinct forces: hydrodynamic (lift and Dean drag) and DEP forces. Moreover, the applied electric fields may generate joule heating, elevating the temperature of the medium, which, in turn, may jeopardize the viability of the cells 38 . Nevertheless, Fig. 9 e indicates that the viability was not considerably changed in the operations of the present study. 5. Conclusion In this study, an inertial-DEP platform with laterally allocated microelectrodes was introduced for the label-free isolation of CTCs from diluted blood samples. To this end, the particle dynamics were first examined in the presence of inertial lift, Dean drag, and DEP forces by the computational-fluid-dynamics simulations. In this regard, a direct numerical simulation was conducted to scrutinize the dynamics. Next, the designed inertial-DEP platform was fabricated using standard soft- and photo-lithography procedures. Then, the performance of the fabricated microfluidic device was tested in solely-inertial and inertial-DEP modes using non-biological and biological samples. As a proof-of-concept, we thoroughly examined the effect of DEP on 5 and 15 µm polystyrene microparticles. It was shown that large non-target micro- and bioparticles can be removed without disrupting the Dean cycle with aid of negative DEP force generated by the microelectrodes. Finally, diluted blood samples spiked with MDA-MB-231 (10 4 CTCs mL − 1 ) cell line were injected into the platform for the separation of MDA-MB-231 cells. Using the proposed spiral-DEP platform, the MDA-MB-231 cells were captured with 89% efficiency at the total flow rate of 650 µL min − 1 without notable changes in viability. While the purity of the isolated CTCs was 85.3% in the absence of the DEP, the purity increased to 94.1% under the voltage of 50 V. Declarations Akownodedgement The authors would like to thank the sharif university of technology research council for the supports. Also, M. 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U.; Zalkovskij, M.; Bilenberg, B.; Bodmer, W.; Kristensen, A.; Marie, R. Separation of cancer cells from white blood cells by pinched flow fractionation. Lab on a Chip 2015, 15 (24), 4598–4606. Au, S. H.; Edd, J.; Stoddard, A. E.; Wong, K. H.; Fachin, F.; Maheswaran, S.; Haber, D. A.; Stott, S. L.; Kapur, R.; Toner, M. Microfluidic isolation of circulating tumor cell clusters by size and asymmetry. Scientific reports 2017, 7 (1), 1–10. Rahmani, A.; Mohammadi, A.; Kalhor, H. R. A continuous flow microfluidic device based on contactless dielectrophoresis for bioparticles enrichment. Electrophoresis 2018, 39 (3), 445–455. Wang, L.; Lu, J.; Marchenko, S. A.; Monuki, E. S.; Flanagan, L. A.; Lee, A. P. Dual frequency dielectrophoresis with interdigitated sidewall electrodes for microfluidic flow-through separation of beads and cells. Electrophoresis 2009, 30 (5), 782–791. Zhao, W.; Zhu, T.; Cheng, R.; Liu, Y.; He, J.; Qiu, H.; Wang, L.; Nagy, T.; Querec, T. D.; Unger, E. R. 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Scientific reports 2013, 3 (1), 1–9. Razavi Bazaz, S.; Rouhi, O.; Raoufi, M. A.; Ejeian, F.; Asadnia, M.; Jin, D.; Ebrahimi Warkiani, M. 3D printing of inertial microfluidic devices. Scientific reports 2020, 10 (1), 1–14. Ghadami, S.; Kowsari-Esfahan, R.; Saidi, M. S.; Firoozbakhsh, K. Spiral microchannel with stair-like cross section for size-based particle separation. Microfluidics and Nanofluidics 2017, 21 (7), 1–10. Mehran, A.; Rostami, P.; Saidi, M. S.; Firoozabadi, B.; Kashaninejad, N. High-Throughput, Label-Free Isolation of White Blood Cells from Whole Blood Using Parallel Spiral Microchannels with U-Shaped Cross-Section. Biosensors 2021, 11 (11), 406. Khan, M.; Chen, X. Numerical study of dielectrophoresis-modified inertial migration for overlapping sized cell separation. Electrophoresis 2022, 43 (7–8), 879–891. Cha, H.; Fallahi, H.; Dai, Y.; Yuan, D.; An, H.; Nguyen, N.-T.; Zhang, J. Multiphysics microfluidics for cell manipulation and separation: a review. Lab on a Chip 2021. Ozkumur, E.; Shah, A. M.; Ciciliano, J. C.; Emmink, B. L.; Miyamoto, D. T.; Brachtel, E.; Yu, M.; Chen, P.-i.; Morgan, B.; Trautwein, J. Inertial focusing for tumor antigen–dependent and–independent sorting of rare circulating tumor cells. Science translational medicine 2013, 5 (179), 179ra147-179ra147. Huang, D.; Xiang, N. Rapid and precise tumor cell separation using the combination of size-dependent inertial and size-independent magnetic methods. Lab on a Chip 2021, 21 (7), 1409–1417. Kim, U.; Oh, B.; Ahn, J.; Lee, S.; Cho, Y. Inertia–acoustophoresis hybrid microfluidic device for rapid and efficient cell separation. Sensors 2022, 22 (13), 4709. Sorour Amini, H.; Mohammadi, A. Microparticle separation using dielectrophoresis-assisted inertial microfluidics: A GPU-accelerated immersed boundary–lattice Boltzmann simulation. Physical Review E 2023, 107 (3), 035307. Zhang, J.; Yuan, D.; Zhao, Q.; Yan, S.; Tang, S.-Y.; Tan, S. H.; Guo, J.; Xia, H.; Nguyen, N.-T.; Li, W. Tunable particle separation in a hybrid dielectrophoresis (DEP)-inertial microfluidic device. Sensors and Actuators B: Chemical 2018, 267 , 14–25. Jeon, H.; Kwon, T.; Yoon, J.; Han, J. Engineering a deformation-free plastic spiral inertial microfluidic system for CHO cell clarification in biomanufacturing. Lab on a Chip 2022, 22 (2), 272–285. Zhou, J.; Papautsky, I. Fundamentals of inertial focusing in microchannels. Lab on a Chip 2013, 13 (6), 1121–1132. Additional Declarations No competing interests reported. Supplementary Files FINALsupp1b.docx Cite Share Download PDF Status: Published Journal Publication published 20 Jul, 2023 Read the published version in Microfluidics and Nanofluidics → Version 1 posted Editorial decision: Major revision 17 Apr, 2023 Reviews received at journal 03 Apr, 2023 Reviewers agreed at journal 30 Mar, 2023 Reviewers invited by journal 30 Mar, 2023 Editor assigned by journal 30 Mar, 2023 Submission checks completed at journal 30 Mar, 2023 First submitted to journal 29 Mar, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2752830","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":187825853,"identity":"bf948177-7d51-4800-a6ab-53cfb2aadf8b","order_by":0,"name":"Aliasghar Mohammadi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7ElEQVRIie3PsWoCQRCA4QkLazOn7clqfAUPQQgh3KucHJyNRcA2RUCwy5Wi4ENYbX1h4VKmFQZEsdXqQEihZNRSWU2XYv9mmv2YWQCX638mz8OITPDA0juAfyeR0YlgdjfJsHkkcJNURiZf/vR0o/yFRfH6tqghiE9CC/Ep6QYfmoKJ8bQa531EkPGzjQD12r6n6WHGRKCMMARsKxtpMKnuNYUzg+sCDxFvqeyspMlE8ZYOE1De8EhQWklASaLqmuKJkWxTJkK2nqYW8khxXt1qekm/DR+2i0IsDVbzje37l4m/PXe5XC7XlX4BUh5IaJo1roIAAAAASUVORK5CYII=","orcid":"","institution":"Sharif University of Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Aliasghar","middleName":"","lastName":"Mohammadi","suffix":""},{"id":187825855,"identity":"6932f826-b6ec-429e-ab1e-aeb5db135cab","order_by":1,"name":"Morteza Safari","email":"","orcid":"","institution":"Sharif University of Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Morteza","middleName":"","lastName":"Safari","suffix":""},{"id":187825856,"identity":"e3f13663-3228-4cf0-b563-ca2b723c2a06","order_by":2,"name":"Mehdi Rahmanian","email":"","orcid":"","institution":"Motamed Cancer Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Mehdi","middleName":"","lastName":"Rahmanian","suffix":""}],"badges":[],"createdAt":"2023-03-29 15:59:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2752830/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2752830/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s10404-023-02662-3","type":"published","date":"2023-07-20T21:41:56+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":35221369,"identity":"4316b99d-d80e-4c44-99a2-a78b5fa3da8a","added_by":"auto","created_at":"2023-04-03 18:52:46","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":373417,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the experimental setup. Syringe pumps I and II were employed for the injection of the blood sample spiked with CTCs and sheath flow, respectively. A generated and amplified electrical voltage was applied to the microelectrodes through copper wires.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/d9be29458360e33b918c5151.png"},{"id":35221367,"identity":"1f606546-1391-41cf-bed3-466b4f7c5cae","added_by":"auto","created_at":"2023-04-03 18:52:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":261707,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic view of the microchannel and microelectrodes together with the trajectory of WBCs in the absence and presence of DEP generated by the microelectrodes.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/0f1bc8734b3a9523aca35864.png"},{"id":35221374,"identity":"f2430227-ca9a-4553-b910-b84aa28da8e5","added_by":"auto","created_at":"2023-04-03 18:52:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":243357,"visible":true,"origin":"","legend":"\u003cp\u003eThe simulated cross-sectional position of the 5 and 15 μm polystyrene microparticles at the outlet of the spiral microchannel at various Dean cycles (a). The simulated distance of the 5 and 15 μm polystyrene microparticles from the inner wall at the outlet as a function of the total flow rate (b). The data in (b) corresponds to the simulation results of (a).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/337d99c8eb74f107cea0c2db.png"},{"id":35222641,"identity":"6b6a06b6-6ece-4d00-91e9-2c1af4a7dfdd","added_by":"auto","created_at":"2023-04-03 19:00:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":485734,"visible":true,"origin":"","legend":"\u003cp\u003eSize distribution of polystyrene microparticles at the inlet (a), inner outlet (b), and outer outlet (c) of the microfluidic device at the total flow rate of 650 μL min\u003csup\u003e-1\u003c/sup\u003e, obtained from the simulation (red lines) and experiments (black lines). Also, the 5 and 15 μm polystyrene microparticles at various cross sections of the spiral microchannel (d), ascertained from the simulation. The injected mixture to the spiral microfluidic device comprised 5 and 15 μm polystyrene microparticles in both the simulation and experiments. The scale bars indicate 60 μm. The size distribution in (a)-(c) were ascertained by the ImageJ software.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/029bf980d7402bb8bd6bb6e6.png"},{"id":35221366,"identity":"c079beef-3d2a-4a64-a106-2e16a32e3089","added_by":"auto","created_at":"2023-04-03 18:52:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":131161,"visible":true,"origin":"","legend":"\u003cp\u003eThe forces at the cross-section of the spiral-DEP platform for 15 μm polystyrene microparticles at the total flow rate of 650 μL min\u003csup\u003e-1\u003c/sup\u003e, voltage of 10 V, and the frequency of 1 MHz. The black-colored arrows indicate the forces. The red-colored dashed circles indicate the equilibrium positions for the 15 μm polystyrene microparticles afforded using the fact that at equilibrium positions \u003cem\u003eF\u003c/em\u003e\u003csub\u003etotal\u003c/sub\u003e = 0.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/a2386c3ba6e37861447562a5.png"},{"id":35222642,"identity":"ef09b9a6-bef6-4081-bb0d-da4a72e98ea4","added_by":"auto","created_at":"2023-04-03 19:00:46","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116097,"visible":true,"origin":"","legend":"\u003cp\u003eSimulated distance of the 15 μm polystyrene microparticle from the inner wall at various voltages as a function of the length in the spiral-DEP platform (a). Simulated cross-sectional position of the 15 μm polystyrene microparticle at the outlet of the spiral-DEP platform (b).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/a0acbaa02a3104b31cf39a63.png"},{"id":35222645,"identity":"0c239f18-86aa-4420-8b29-73f6fcae09ac","added_by":"auto","created_at":"2023-04-03 19:00:47","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":332646,"visible":true,"origin":"","legend":"\u003cp\u003eExperimentally measured size distribution of the 15 μm polystyrene microparticles at the inlet, inner outlet, and outer outlet of the spiral-DEP platform at the total flow rate of 650 μL min\u003csup\u003e-1\u003c/sup\u003e as a function of the applied voltage.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/6f4bee1fdade6c31f2b13eae.png"},{"id":35223791,"identity":"4c5b06fd-0328-450d-9421-2781acf5f5c6","added_by":"auto","created_at":"2023-04-03 19:08:46","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":586733,"visible":true,"origin":"","legend":"\u003cp\u003eSimulated distance of the 5 μm polystyrene microparticles from the inner wall at various voltages as function of the length in the spiral-DEP platform (a). Simulated lateral position of the 5 μm polystyrene microparticles at the outlet of the spiral-DEP platform at various voltages (b). Particle stream width at different voltages ascertained by image processing using ImageJ software (c). Microscopic images from the 5 μm polystyrene particlesin the outer region of the spiral-DEP platform at 0, 20, and 50 V (d). The scale bars indicate 100 μm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/3d82a88e6dd29bf884e73f6c.png"},{"id":35221371,"identity":"fee8c5b8-8c18-44f0-bd12-965d27d98260","added_by":"auto","created_at":"2023-04-03 18:52:46","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":611625,"visible":true,"origin":"","legend":"\u003cp\u003eThe rejection ratio as a function of the total flow rate for the diluted blood samples (a). The influence of the blood Hct on the blood cell stream width at the outlet from the spiral microchannel (b). The collected samples from the inner and outer outlets of the spiral microchannel for the sample and sheath flow rates of, respectively, 100 and 550 μL min\u003csup\u003e-1\u003c/sup\u003e (c). The effect of the voltage on the separation purity (d). The viability of the MDA-MB-231 cells before and after injection to the spiral-DEP platform (e). Fluorescence images from the inner and outer outlets in which the blue-colored dots are MDA-MB-231 cells, stained with DAPI (f). Bright-field images from the inner and outer outlets (g). The scale bars are 139, 25, and 25 µm in, respectively, (f), (g), and. All the plots represent the mean value and the standard deviation (n = 3 and p-value = 0.05).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/a075200d2d57c0f5c323ca94.png"},{"id":44735445,"identity":"7188715d-d643-4ed0-b8c8-01cc646e524c","added_by":"auto","created_at":"2023-10-16 22:25:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3249320,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/cf89a514-f697-4ef9-9e5e-7bc2ab4de12b.pdf"},{"id":35222643,"identity":"eeaa0e8e-33fb-4176-9905-18012c408ef0","added_by":"auto","created_at":"2023-04-03 19:00:46","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":3199098,"visible":true,"origin":"","legend":"","description":"","filename":"FINALsupp1b.docx","url":"https://assets-eu.researchsquare.com/files/rs-2752830/v1/88ce11d318ddca20b6c86922.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Label-free isolation of circulating tumor cells using negative lateral dielectrophoresis- assisted inertial microfluidics","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eDespite the increasing clinical advances in cancer diagnosis and treatment, cancer has remained one of the deadliest diseases in the world \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The high mortality rate of cancer arises from the aggressive spread of this disease throughout the body through a phenomenon known as metastasis \u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Metastasis is responsible for more than 90% of cancer mortality. As a matter of course, metastasis is the most important barrier in our combat against cancer; such that cancer mortality does not decrease unless using a reliable biomarker to diagnose metastasis at the early stages \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn the process of metastasis, circulating tumor cells (CTCs) are shed from the primary tumor and conveyed to distant organs via circulation \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Therefore, CTCs play a pivotal role in the development of secondary tumors \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The vitality of metastasis is more clarified when we consider that a 1-g tumor releases more than two million CTCs per day \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. However, CTCs have a limited half-life and many of them fail to survive the physical tensions in the bloodstream \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Nevertheless, few detached survived CTCs can spread cancer by generating tumors in other organs \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Apart from the formation of secondary tumors, the lack of a unique prescription for all patients due to the heterogeneity of tumor cells is another challenge, which increases cancer mortality. In this regard, personalized therapy can lead to a shorter and more effective treatment period \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe current approaches for cancer diagnosis hinge upon invasive procedures such as tissue and bone biopsy. In this context, enumeration and isolation of CTCs can be an alternative strategy for early cancer diagnosis in a less invasive manner. Moreover, this approach provides a real-time assessment of a patient condition as it simply demands a blood sample or other bodily fluids \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. In other words, the number of CTCs in the blood could be an indicator of the tumor response to a specific therapy \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, capturing viable CTCs provides the opportunity to conduct in vitro studies for the development of anti-cancer drugs \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the isolation of CTCs is challenging due to their low concentration and size, which overlaps those of white blood cells (WBCs) \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eConventional techniques for the isolation of CTCs include density-gradient centrifugation, flow cytometry, and fluorescence-activated as well as magnetic-activated cell sorting. Even though such techniques are well established, they require bulky and expensive equipment as well as detailed protocols. On the other hand, microfluidic-based techniques provide the opportunity to regulate and process a small amount of sample within a short time at a lower cell separation cost. As such, microfluidics has increasingly come into focus for cell separation and manipulation \u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn general, microfluidic-based cell separation techniques include two groups of passive and active. Passive techniques rely on differences among some intrinsic properties of cells, such as size, shape, and deformability \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. On the other hand, active techniques use external forces for separating cells upon differences in dielectric properties, magnetic susceptibility, compressibility, or refractive index \u003csup\u003e\u003cspan additionalcitationids=\"CR22 CR23 CR24\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAmong the passive techniques, inertial-based separations have been in focus for the separation of cancer cells in the last two decades (see section S1.1 of the supporting information for a brief introduction to the inertial migration in microchannels) \u003csup\u003e\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. In this context, various microchannels with diverse geometries were reported for the improved isolation of CTCs \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan additionalcitationids=\"CR31 CR32 CR33 CR34 CR35\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In spite of this fact, the contamination of isolated CTCs with WBCs is inevitable in size-based inertial microfluidics separations, due to the overlapped sizes of CTCs and WBCs \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e (see section S1.2 of the supporting information for a brief introduction to WBCs). Moreover, the separation of cells is not easily regulated in inertial-based methods once the microfluidic device geometry is fixed.\u003c/p\u003e \u003cp\u003eKeeping this in mind, active methods can be utilized for separating same-sized cells, where the separation performance can be modulated through tuning externally controllable parameters. Nevertheless, active methods are a proper choice for cell separation at limited throughput \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThus, hybrid platforms have been developed for cell separation. A combination of the high precision of active methods and the high throughput of inertial-based methods can result in a powerful cell separation platform. Among the previously reported hybrid systems \u003csup\u003e\u003cspan additionalcitationids=\"CR40 CR41\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e, synergizing inertial-based methods with dielectrophoresis (DEP) appears a proper combination (see section S1.3 of the supporting information for a brief introduction to the dielectrophoresis). In this venue, of the studies reported as the inertial-DEP combination was a hybrid inertial-DEP platform \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, where the DEP was introduced through full coupling with a serpentine microchannel. A sheath flow was used to eliminate the upper inertial equilibrium positions. Then, the DEP force was competing with the inertial lift force along the vertical direction, levitating particles in the vertical direction. Subsequently, differential focusing positions of particles along the lateral direction were attained. The inertial-DEP platform was, however, demanding a careful selection of operational parameters to modulate the equilibrium positions. For instance, the positions were laterally differentiated at specific voltages; otherwise, no lateral differential focusing was observed. As such, there is still a lack of an inertial-DEP platform to easily control the equilibrium positions along the lateral direction.\u003c/p\u003e \u003cp\u003eIn this study, a spiral microchannel was coupled with laterally allocated microelectrodes to form a, so-called, spiral-DEP platform (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The developed platform had many advantages over the current microfluidics particle manipulation methods. This method was based on negative dielectrophoresis such that particles were not exposed to the strong electric fields near the microelectrodes. This is especially important for bioparticles, where prolonged exposure to strong electric fields leads to cell damage. In this context, the developed platform was used to isolate CTCs from blood. The designed microelectrodes were responsible for the deflection of the non-target cells towards the outer wall based on a negative DEP force as well as the crossover frequencies of different bioparticles. All types of non-target cells were removed, irrespective of their size-overlap with target cells, by taking advantage of the selective inertial focusing for the inertial section and negative DEP for the dielectrophoresis section. Thus, WBC contamination was avoided in a label-free approach.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe remainder of this study is laid out as follows. Section 2 outlines the design and simulation of the microfluidic device. Fabrication procedures and experiments are discussed in section \u003cspan refid=\"Sec3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Results are provided and discussed in section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Finally, a summary is given in section \u003cspan refid=\"Sec8\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e"},{"header":"2. Design And Simulation Of The Microfluidic Device","content":"\u003cp\u003eIn this study, a spiral microchannel with three turns as well as two inlets and two outlets was employed. The inner radius and distance between the turns of the spiral microchannel were, respectively, 6 and 1 mm. Moreover, the width and depth of the microchannel were 500 and 170 \u0026micro;m, respectively. The inner and outer inlets were employed for the injection of the sample and sheath fluid, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The sample was diluted CTC-spiked blood while the sheath fluid was phosphate-buffered saline (PBS). In addition, CTCs and blood cells were to come out of the microchannel through the inner and outer outlets, respectively. In other words, the CTCs and the blood cells are the target and non-target cells, respectively.\u003c/p\u003e \u003cp\u003eAlthough many of the non-target cells are to remove in the spiral microchannel using the Dean-cycle phenomenon (see section S1.1 of the supporting information), such microfluidic devices fail to remove non-target cells whose size overlaps the size of the target cells. In the developed spiral microchannel, CTCs (target) and large WBCs (non-target) focus close to the inner wall, reducing the purity of the isolated CTCs. To increase the purity of the isolated CTCs, the DEP force was employed to push WBCs away from the inner wall. In this venue, the conventional practice to generate such negative DEP force involves embedding microelectrodes on one side of the microchannel. However, this approach was challenging due to geometrical restrictions. As such, an unprecedented microelectrode pattern (figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) was designed to generate the negative DEP force to repel non-target cells from the inner wall. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the Dean drag and lift forces cancel out each other at the equilibrium positions, such that the microelectrode-generated negative DEP force pushes the cells away from the inner wall.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe proper selection of operational parameters solely through experimental tests is time-consuming and costly due to the complex nature of cell dynamics resulting from the inertial lift, Dean drag, and DEP forces. Accordingly, numerical simulations were included to assist in the design of the microfluidic device. In this regard, the details of the simulation procedure are provided in section S2 of the supporting information.\u003c/p\u003e \u003cp\u003eBriefly, a three-dimensional direct numerical simulation was conducted to examine the effect of inertial lift, Dean drag, and DEP forces on the micro- and bioparticle dynamics in the spiral-DEP platform. For this purpose, the COMSOL Multiphysics software version 5.6 was used to build a model for solving fluid flow and electric field. Then, the simulated fluid flow and electric field were used to calculate the hydrodynamic (Dean drag, added mass, and lift forces) and DEP forces for the particle tracing analysis of the micro- and bioparticles.\u003c/p\u003e"},{"header":"3. Microfluidic Device Fabrication And Operating Procedures","content":"\u003cp\u003eThe proposed microfluidic device was comprising two parts: the microchannel and the microelectrodes. While the former was fabricated by standard soft-lithography procedure, the microelectrodes were prepared by conventional photolithography. The fabrication procedures are described in section S8 of the supporting information.\u003c/p\u003e \u003cp\u003eTwo types of experiments were performed with the spiral-DEP platform. In the earlier experiments, the physics of solely-inertial as well as inertial-DEP microfluidics was examined by separating polystyrene microparticles. To this end, a binary mixture of 5 and 15 \u0026micro;m polystyrene microparticles was suspended in deionized water at a volume fraction of 0.1 vol.%. In the latter experiments, the isolation of CTCs was addressed using the spiral-DEP platform with diluted blood samples spiked with the MDA-MB-231 cell line such that the resulting concentration of the CTCs within the diluted blood samples was 10\u003csup\u003e4\u003c/sup\u003e CTCs mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe details of the procedures used for the preparation of the non-biological and biological samples are described in section S9 of the supporting information. Also, prepared samples were injected into the microfluidic device based on the operating procedure described in section S10 of the supporting information. It must be noted that throughout all the experiments, the sample flow rate was fixed at 100 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the sheath fluid flow rate ranged from 150 to 750 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eMeanwhile, three measures were used for the interpretation of the resulting data and assessing the microfluidic device performance:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\text{E}\\text{f}\\text{f}\\text{i}\\text{c}\\text{i}\\text{e}\\text{n}\\text{c}\\text{y}=\\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{t}\\text{a}\\text{r}\\text{g}\\text{e}\\text{t}\\text{e}\\text{d} \\text{p}\\text{a}\\text{r}\\text{t}\\text{i}\\text{c}\\text{l}\\text{e}\\text{s}/\\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{n}\\text{e}\\text{r} \\text{o}\\text{u}\\text{t}\\text{l}\\text{e}\\text{t}}{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{t}\\text{a}\\text{r}\\text{g}\\text{e}\\text{t}\\text{e}\\text{d} \\text{p}\\text{a}\\text{r}\\text{t}\\text{i}\\text{c}\\text{l}\\text{e}\\text{s}/\\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{l}\\text{e}\\text{t}}, \\left(1\\right)$$\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{P}\\text{u}\\text{r}\\text{i}\\text{t}\\text{y}=\\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{t}\\text{a}\\text{r}\\text{g}\\text{e}\\text{t}\\text{e}\\text{d} \\text{p}\\text{a}\\text{r}\\text{t}\\text{i}\\text{c}\\text{l}\\text{e}\\text{s}/\\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{n}\\text{e}\\text{r} \\text{o}\\text{u}\\text{t}\\text{l}\\text{e}\\text{t}}{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{p}\\text{a}\\text{r}\\text{t}\\text{i}\\text{c}\\text{l}\\text{e}\\text{s}/\\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{n}\\text{e}\\text{r} \\text{o}\\text{u}\\text{t}\\text{l}\\text{e}\\text{t}}, \\left(2\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eand\u003cdiv id=\"Equc\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equc\" name=\"EquationSource\"\u003e\n$$\\text{R}\\text{e}\\text{j}\\text{e}\\text{c}\\text{t}\\text{i}\\text{o}\\text{n} \\text{r}\\text{a}\\text{t}\\text{i}\\text{o}=1-\\frac{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{b}\\text{l}\\text{o}\\text{o}\\text{d} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{n}\\text{e}\\text{r} \\text{o}\\text{u}\\text{t}\\text{l}\\text{e}\\text{t}}{\\text{n}\\text{u}\\text{m}\\text{b}\\text{e}\\text{r} \\text{o}\\text{f} \\text{b}\\text{l}\\text{o}\\text{o}\\text{d} \\text{c}\\text{e}\\text{l}\\text{l}\\text{s} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{i}\\text{n}\\text{p}\\text{u}\\text{t} \\text{b}\\text{l}\\text{o}\\text{o}\\text{d} \\text{s}\\text{a}\\text{m}\\text{p}\\text{l}\\text{e}}. \\left(3\\right)$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eMoreover, a trypan blue exclusion assay was performed in solely-inertial and inertial-DEP platforms to measure the viability of the CTCs \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e"},{"header":"4. Results And Discussion","content":"\u003cp\u003eThe aim of this study is to improve the purity of the CTCs isolation from blood utilizing the synergistic effect of the inertial and dielectrophoresis phenomena. In this regard, the dynamics of different-sized polystyrene microparticles was first simulated to determine their lateral positions at the outlet of the spiral microfluidic device. In the second part of this section, the dielectrophoretic force was applied to manipulate the lateral positions of the microparticles at the outlet. Finally, in the last part of this section, the isolation of the CTCs from blood was experimentally investigated to examine the robustness of the spiral-DEP microfluidic device.\u003c/p\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e4.1. Dynamics of microparticles in the spiral microfluidic device\u003c/h2\u003e \u003cp\u003eThe dynamics of polystyrene microparticles, with the sizes of 5 and 15 \u0026micro;m, in the spiral microfluidic device was simulated at various flow rates. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e depicts the distance of the microparticles from the inner wall at the outlet of the spiral microfluidic device. It is observed that the total flow rate considerably modulates the distance of all the particles.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThis observation is explained by considering the forces governing the dynamics of the microparticles. At the entrance region of the spiral microfluidic device, the particles were focused close to the outer wall with the aid of a sheath flow. In such a situation, the particle motion is governed by the competition between the inertial lift force \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mathbf{F}}_{\\text{l}\\text{i}\\text{f}\\text{t}}\\)\u003c/span\u003e\u003c/span\u003e and the drag force exerted by the Dean vortices \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mathbf{F}}_{\\text{D}\\text{e}\\text{a}\\text{n}}\\)\u003c/span\u003e\u003c/span\u003e. Due to the different scaling of these forces \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left|{\\mathbf{F}}_{\\text{l}\\text{i}\\text{f}\\text{t}}\\right| \\tilde{ \\text{d}}_{\\text{p}}^{4}\\)\u003c/span\u003e\u003c/span\u003e and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left|{\\mathbf{F}}_{\\text{D}\\text{e}\\text{a}\\text{n}}\\right| \\tilde{\\text{d}}_{\\text{p}}\\)\u003c/span\u003e\u003c/span\u003e, the microparticles undergo two different trajectories based on their sizes. For the larger particles, both the forces are relevant. At the central region close to the inner wall, the force field resulting from their superposition brings the larger particles to two equilibrium positions. In spite of this fact, all the larger particles should have time to reach the equilibrium positions. As such, there is a lower limit for the total flow rate to inertially focus the larger particles. This fact is apparent in the focusing of the larger particles, shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. The fosuing position of the larger particles changes with increasing the total flow rate.\u003c/p\u003e \u003cp\u003eThe dynamics of the smaller particles is, however, modulated by the Dean drag forces rather than the lift forces. In this extreme, the smaller particles follow two symmetrical vortices, remaining randomly distributed. Since the fluid circulates laterally due to the vortices, the smaller particles are in a continuous lateral circulation. Thus, the positions of the smaller particles at the outlet rely on the geometry of the microchannel and the flow rate. For a spiral microchannel with specified geometry, the positions are solely subject to the flow rate. The positions of the smaller particles oscillates as a function of the flow rate (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). This fact modulates the separation purity for the larger particles. In order to afford the highest separation purity, the total flow rate must be at specific values to ensure an integer number of, so-called, Dean cycle (see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eConsidering the aforementioned two constarints for the proper flow rate, we opted the total flow rate of 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to provide both complete inertial migration and 1 Dean cycle. It must be noted that the total flow rate of 1300 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e satisfies the aforementioned constraints, but we selected 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e as the opted total flow rate. As PDMS deforms with increasing the flow rate, leading to unreliable results owing to changing the dimensions and shape of the microchannels cross-section \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAfter selecting the proper flow rates, the sepration of the 5 and 15 \u0026micro;m polystyrene microparticles using the spiral microfluidic device was simulated, and compared with the experimental results. The separation results from the simulation and experiments are shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec. It was experimentally obtained that the polystyrene microparticles were separated with the efficiency and purity of 78% and 96.1%, respectively. This finding indicates that the opted flow rates were proper such that the larger particles migrate inertially towards the inner wall while the smaller particles circulate laterally towards the outer wall. Furthermore, our computational results not only confirm this phenomenon but also demonstrate that inertial migration occurs in two stages (see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), where the larger particles migrate first to an equilibrium perimeter and slowly to the center of the faces \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Dynamics of microparticles in the spiral-DEP microfluidic device\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e demonstrates the contributing forces on the dynamics of the 15 \u0026micro;m polystyrene microparticles in the spiral-DEP platform. The equilibrium positions of the microparticles are calculated using the constraint \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\({\\mathbf{F}}_{\\text{t}\\text{o}\\text{t}\\text{a}\\text{l}}=0\\)\u003c/span\u003e\u003c/span\u003e. It is observed that the equilibrium positions of the 15 \u0026micro;m polystyrene microparticles are modulated in the presence of the DEP force. The real part of the Clausius-Mossotti factor determines the direction of the DEP force. In this context, the real part of the Clausius-Mossotti factor is negative for the 15 \u0026micro;m particles at the frequency of 1 MHz, and the DEP force is directed away from the microelectrodes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, the equilibrium position corresponding to the lower wall is considerably displaced, while the equilibrium position corresponding to the top wall remains almost intact by the DEP force generated from the microelectrodes. The reason is the short-range nature of the DEP force, which exponentially decays by the distance from the microelectrodes.\u003c/p\u003e \u003cp\u003eWe simulated the dynamics of the 15 \u0026micro;m polystyrene microparticles in the presence of the DEP force. In the spiral-DEP platform, there is no allocated microelectrodes within the initial 1 cm of the spiral microchannel; as such, the lateral position of the microparticles does not change by increasing the voltage within the initial 1 cm. Also, the curvature is less in the initial first turn of the spiral microchannel; thus, the effect of the Dean drag force is significant and the position of the microparticles does not change with varying the voltage in the first 3 cm of the spiral microchannel (region #1 in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). In the subsequent region, the 15 \u0026micro;m polystyrene microparticles are gradually directed away from the microelectrodes in the presence of the DEP force (region #2 in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). It is observed that a negative DEP force directs away particles from inertial equilibrium positions in the vicinity of the microelectrodes. In this venue, the effect of the DEP force was experimentally examined on the dynamics of the 15 \u0026micro;m polystyrene microparticles. To this end, a sample of the 15 \u0026micro;m polystyrene microparticles was injected into the spiral-DEP microfluidic device at a total flow rate of 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Meanwhile, the frequency of the applied electric field was set 1 MHz and the voltage increased from 0 to 50 V.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e depicts the size distribution of the microparticles at the inlet and outlets of the spiral-DEP microfluidic device. The majority of the microparticles (⁓96%) migrated towards the inner wall in the absence of the DEP force, whereas the microparticles start to migrate towards the outer wall with increasing the voltage as a result of the negative DEP force generated by the microelectrodes (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec). This indicates that the microelectrodes enable repelling non-target micro- and bioparticles. However, given movie S1 and Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec, the microparticles did not completely leave through the outer wall as the equilibrium position corresponding to the top wall remains almost intact by the DEP force generated from the microelectrodes (see figure S7).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurthermore, the effect of the DEP force on the dynamics of the 5 \u0026micro;m polystyrene microparticles was computationally and experimentally investigated (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). It is observed that the DEP force does not disrupt the Dean cycle and the 5 \u0026micro;m particles migrate to the outer wall (1 Dean cycle) at the total flow rate of 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo understand the physics underlying this observation, recall the scaling of \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\left|{\\mathbf{F}}_{\\text{D}\\text{E}\\text{P}}/{\\mathbf{F}}_{\\text{D}\\text{e}\\text{a}\\text{n}}\\right| \\tilde{ \\text{d}}_{\\text{p}}^{3}\\)\u003c/span\u003e\u003c/span\u003e (see equations S4 and S5). Accordingly, the dynamics of the 5 \u0026micro;m particles is modulated by the Dean drag force rather than the DEP force. This indicates that the 5 \u0026micro;m particles are driven by the Dean vortices. We, however, note that the position of the 5 \u0026micro;m microparticles slightly changes with increasing the voltage (see Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). The position of the 5 \u0026micro;m polystyrene microparticles does not notably change until the second turn (region #1), but they, in the third turn, start to migrate towards the outer wall with increasing the voltage (region #2). This fact is associated with the gradual increase of the curvature radius in the spiral microchannel, where the magnitude of the Dean drag force in the third turn is not as strong as that in the first and second turns. Thus, the 5 \u0026micro;m microparticles were slightly pushed towards the outer wall owing to the negative DEP force generated by the microelectrodes. This phenomenon results in the decrement of the width of the microparticle stream at the outlet, which improves the separation purity (see Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ec and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Spiral-DEP platform in the context of CTCs isolation\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ea shows the measured rejection ratio for the diluted blood samples at two distinct hematocrit levels of 1 and 10% as a function of the total flow rate. The highest rejection ratio was recorded at the total flow rate of ~\u0026thinsp;650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for the considered hematocrit levels. As also revealed by the plots in the figure, the rejection ratio decreases with increasing the hematocrit level. Needless to say, separating the blood cells from the serum is more difficult at higher hematocrits.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the next step, a diluted blood sample at the hematocrit level of 1% was spiked with the MDA-MB-231 cell line and injected into the spiral-DEP platform to separate the CTCs from the blood sample. The influence of the voltage on the separation purity is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ed. The purity shows an ascending trend by increasing the voltage. The impact of the DEP becomes imperative with increasing the voltage. This observation is associated with the forces contributing to the cell dynamics in the DEP-spiral platform, which are the inertial lift, Dean drag, and DEP forces. At zero voltage, the CTCs were isolated under the influence of hydrodynamic forces that distinguish cells based on their size. In this case, the purity of the MDA-MB-231 cell line was 85.3%. With increasing the voltage, the DEP force becomes imperative for the bioparticle migration. Therefore, the purity increased to 94.1%. This is mainly because the negative DEP force generated by the microelectrodes contributes to removing larger WBCs that the spiral microfluidic device could not remove.\u003c/p\u003e \u003cp\u003eLet us continue with the task of addressing the impact of the DEP force on the separation efficiency of the CTCs. While the efficiency was found 88.0% in the absence of DEP, the efficiency was 89.0% for the total flow rate of 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and the applied voltage of 50 V. Therefore, the DEP does not have a considerable effect on efficiency. This fact arises from the selected frequency at which the CTCs experience a relatively small DEP force.\u003c/p\u003e \u003cp\u003eFinally, the effect of the cell separation by the spiral-DEP platform on the viability of the isolated CTCs was explored. During the operation, the cells experience stresses from two distinct forces: hydrodynamic (lift and Dean drag) and DEP forces. Moreover, the applied electric fields may generate joule heating, elevating the temperature of the medium, which, in turn, may jeopardize the viability of the cells \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e. Nevertheless, Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003ee indicates that the viability was not considerably changed in the operations of the present study.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn this study, an inertial-DEP platform with laterally allocated microelectrodes was introduced for the label-free isolation of CTCs from diluted blood samples. To this end, the particle dynamics were first examined in the presence of inertial lift, Dean drag, and DEP forces by the computational-fluid-dynamics simulations. In this regard, a direct numerical simulation was conducted to scrutinize the dynamics. Next, the designed inertial-DEP platform was fabricated using standard soft- and photo-lithography procedures. Then, the performance of the fabricated microfluidic device was tested in solely-inertial and inertial-DEP modes using non-biological and biological samples. As a proof-of-concept, we thoroughly examined the effect of DEP on 5 and 15 \u0026micro;m polystyrene microparticles. It was shown that large non-target micro- and bioparticles can be removed without disrupting the Dean cycle with aid of negative DEP force generated by the microelectrodes. Finally, diluted blood samples spiked with MDA-MB-231 (10\u003csup\u003e4\u003c/sup\u003e CTCs mL\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) cell line were injected into the platform for the separation of MDA-MB-231 cells. Using the proposed spiral-DEP platform, the MDA-MB-231 cells were captured with 89% efficiency at the total flow rate of 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e without notable changes in viability. While the purity of the isolated CTCs was 85.3% in the absence of the DEP, the purity increased to 94.1% under the voltage of 50 V.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAkownodedgement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors would like to thank the sharif university of technology research council for the supports. Also, M. Safari would like to appreciate MEMS RASA company for technical support in the fabrication of the microfluidic device.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors state that there is no conflict of interest.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSiegal, R.; Miller, K. D.; Jemal, A. Cancer statistics, 2012. Ca Cancer J Clin 2014, \u003cem\u003e64\u003c/em\u003e (1), 9\u0026ndash;29.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSleeboom, J. 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Lab on a Chip 2013, \u003cem\u003e13\u003c/em\u003e (6), 1121\u0026ndash;1132.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microfluidics-and-nanofluidics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mano","sideBox":"Learn more about [Microfluidics and Nanofluidics](http://link.springer.com/journal/10404)","snPcode":"10404","submissionUrl":"https://submission.nature.com/new-submission/10404/3","title":"Microfluidics and Nanofluidics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-2752830/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2752830/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn this study, a dielectrophoresis (DEP)-assisted inertial microfluidics methodology was introduced for the the isolation of circulating tumor cells (CTCs) from diluted blood samples. The methodology was based on the negative DEP, provided with the laterally allocated microelectrodes as well as the proper selection of the applied electric field frequency and voltage, to suppress the limited purity arising from the overlapped sizes of CTCs and white blood cells. Initially, the dynamics of 5 and 15 \u0026micro;m polystyrene microparticles within the DEP-assisted inertial microfluidic device were numerically and expimentally investigated. While the dynamics of the larger microparticles was governed by the inertial and DEP forces, those of the smaller microparticles were subject to the Dean drag force. In the absence of the DEP force, the larger microparticles migrate to two stable equilibrium positions corresponding to the upper and lower walls for the microchannel cross-section. In the presence of the DEP force, the equilibrium position corresponding to the lower wall is considerably displaced, while the equilibrium position corresponding to the top wall remains almost intact. Finally, it was found that the methodology outperformed the corresponding solely-inertial methodology in terms of purity for the isolation of CTCs from diluted blood samples. For instance, the purity of isolated MDA-MB-231 spiked in diluted blood samples, at a hematocrit of 1%, by the solely-inertial microfluidic device was 85.3%, while viable CTCs were captured using the DEP-assisted inertial microfluidic device with 94.1% purity at the total flow rate and applied voltage of, respectively, 650 \u0026micro;L min\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 50 V.\u003c/p\u003e","manuscriptTitle":"Label-free isolation of circulating tumor cells using negative lateral dielectrophoresis- assisted inertial microfluidics","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-04-03 18:52:42","doi":"10.21203/rs.3.rs-2752830/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-04-17T18:27:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-04-03T09:14:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84e7096a-690c-43ac-988f-0d3fd8be992a","date":"2023-03-30T13:35:18+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-03-30T13:24:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-03-30T13:16:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-03-30T13:07:50+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microfluidics and Nanofluidics","date":"2023-03-29T15:49:04+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microfluidics-and-nanofluidics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mano","sideBox":"Learn more about [Microfluidics and Nanofluidics](http://link.springer.com/journal/10404)","snPcode":"10404","submissionUrl":"https://submission.nature.com/new-submission/10404/3","title":"Microfluidics and Nanofluidics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1b861873-4e40-4faf-bdfa-79747eae8a6f","owner":[],"postedDate":"April 3rd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-10-16T22:09:43+00:00","versionOfRecord":{"articleIdentity":"rs-2752830","link":"https://doi.org/10.1007/s10404-023-02662-3","journal":{"identity":"microfluidics-and-nanofluidics","isVorOnly":false,"title":"Microfluidics and Nanofluidics"},"publishedOn":"2023-07-20 21:41:56","publishedOnDateReadable":"July 20th, 2023"},"versionCreatedAt":"2023-04-03 18:52:42","video":"","vorDoi":"10.1007/s10404-023-02662-3","vorDoiUrl":"https://doi.org/10.1007/s10404-023-02662-3","workflowStages":[]},"version":"v1","identity":"rs-2752830","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2752830","identity":"rs-2752830","version":["v1"]},"buildId":"FbvkV6FR0MCFSLy54lSbu","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}