A multilayer polymer-film inertial microfluidic device for high

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A multilayer polymer-film inertial microfluidic device for high-throughput cell concentration Nan Xiang, Rui Zhang, Yu Han, and Zhonghua Ni Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b01116 • Publication Date (Web): 28 Mar 2019 Downloaded from http://pubs.acs.org on March 28, 2019

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Analytical Chemistry

A multilayer polymer-film inertial microfluidic device for high-throughput cell concentration Nan Xiang*, Rui Zhang, Yu Han and Zhonghua Ni* School of Mechanical Engineering, and Jiangsu Key Laboratory for Design and Manufacture of Micro-Nano Biomedical Instruments, Southeast University, Nanjing, 211189, China. *E-mails: [email protected]; [email protected]; Phone: +86-025-52090518. Fax: +86-025-52090501. ORCID: Nan Xiang 0000-0001-9803-4783 Abstract We report here a novel multilayer polymer-film inertial microfluidic (MPIM) device with complex three dimensional (3D) fluidic paths for realizing the massive parallelization of multiplexing functional channels. The proposed MPIM device is fabricated by simply stacking different polymer-film channel layers and adhesive layers. As a prototype demonstration, a MPIM device with multiplexing radially-arrayed serpentine channels is designed and fabricated. Seven functional layers are stacked in vertical direction to create the 3D fluidic paths for realizing the functions of inertial cell concentration, fluid converging and flow resistance adjustment in an all-in-one device with a thickness of only 1.4 mm. Then, the physics and the concentration performances of our MPIM device are explored. The results indicate that our MPIM device is capable of concentrating various particles at a throughput up to 8 ml/min. Finally, we successfully apply our MPIM device for the high-throughput concentration of the microalgae and trace tumor cells from large background fluids. We envision the wide application of our MPIM device as a low-cost “on-chip centrifuge” for the concentration of various bioparticles.

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Introduction Inertial microfluidics has attached increasing interests in the fields of disease diagnosis, environment monitoring and biomedical research due to its numerous advantages such as label-free operation, simple structure, and high processing throughput.1,2 As a new promising technique for engineering the cells or fluids at microscale, inertial microfluidics has been widely applied for sample pre/posttreatment (e.g., focusing3,4, separation5,6, concentration7,8, filtration9 and trapping10), continuous-flow cell detection (e.g., microflow cytometer11, real-time cell mechanotyping12), single-cell analysis (e.g., droplet encapsulation13) and microparticle fabrication14 over the past years. In addition to these novel applications, the physics behind the inertial flows coupled with or without Dean vortex or viscoelasticity effects has been explored in previous studies.4,15-19 To make the inertial microfluidic device commercially viable, the mass production of disposable devices in a low-cost manner is especially important. However, most of the previously-reported inertial microfluidic devices are still made of poly(dimethylsiloxane) (PDMS) which is mainly employed for research use and does not translate well to a commercial scale. Recently, Papautsky et al.20 applied the rollto-roll (R2R) hot embossing to develop a disposable thermoplastic straight channel device with a low aspect-ratio for size-based sorting. The R2R hot embossing is a potential method for the mass production of inertial microfluidic devices. However, the cross-section of the fabricated channel shows a trapezoid shape and this method is incapable of creating complex channels with three dimensional (3D) fluidic paths. We developed a “syringe filter” like inertial microfluidic device using the 3D printing for hand-powered cell concentration.8 However, the 3D printing is still challenge to print long complex microchannels with well-controlled cross-sectional shapes. Paiè et al.21 employed the femtosecond laser irradiation followed by chemical etching to create a spatial serpentine channel for achieving particle focusing. However, the etching of long channels is very challenge and time consuming. Other unconventional methods for fabricating inertial microfluidic devices include the rolling of tubes in the spiral layout.22,23 Another important consideration for accelerating the commercial transformation of inertial microfluidics is the multiplexing channel massive parallelization which increases the processing throughput of inertial microfluidics to meet the requirement of practical applications. Up to now, researchers have made continuous attempts to parallelize the inertial microfluidic channel in a radial or array layout. For example, Warkiani et al.24,25 proposed a multiplexing spiral channel device for blood plasma isolation or cell cycle synchronization through vertically stacking thick PDMS layers with multiplex channels. As the channel layers are simply stacked, the samples will unequally distribute into different layers when the layer number is large. In addition, the footprints of these devices are very large because the spiral is difficult to be parallelized. The straight and serpentine channels can be more easily parallelized as compared with spiral channels. Di Carlo et al.26 developed a massively parallel blood filtration device with 40 radially-arrayed straight channels for filtering blood plasma at 2


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a flow rate of 8 ml/min. However, this massively parallel device has a large footprint of 7 cm×7 cm and 120 outlets. They also developed another device containing 256 high aspect-ratio parallel channels as a particle pre-focusing unit for the image-based flow cytometer.27 Although up to 256 channels were parallelized, the flow-rate throughput was limited to 2.5 ml/min due to the small channel cross-sections. Zhang et al.28 proposed a device with 8 parallelized serpentine channels for the extraction of blood plasma at a flow-rate throughput of 2.8 ml/min. Martel et al.7 developed a serpentine channel with multiplex micro-siphoning units and they parallelized these channels for continuous cell concentration at a flow-rate throughput of 4 ml/min. However, the channel structure is very complex as a careful flow-resistance control of each microsiphoning unit is required. In addition, the fabrication of these large PDMS devices via soft lithography is rather time-consuming and any defect in the photolithographyfabricated master will make the PDMS replicas invalid. Herein, we propose a novel multilayer polymer-film inertial microfluidic (MPIM) device with complex 3D fluidic paths through vertically stacking different polymerfilm channel layers and adhesive layers. As a prototype demonstration, an all-in-one MPIM device with multiplexing radially-arrayed serpentine channels but with only one inlet and two outlets are designed for achieving cell concentration. To understand the physics of our device, the inertial focusing dynamics in a single channel is explored. Then, the concentration performances of our MPIM device are characterized over a wide flow-rate range. Finally, we apply our MPIM device for achieving the highthroughput concentration of microalgae and trace tumor cells from a large-volume background fluid. The developed MPIM device offers special advantages of simplicity in fabrication, complexity in fluidic capability and extremely low cost, and may provide important insights for developing the next generation microfluidic centrifuge.

Materials and Methods Conceptual design As illustrated in Figure 1(a), our MPIM device consists of seven functional layers including one parallel channel layer (#1), three adhesive layers (#2, #4 and #6), two confluence channel layers (#3 and #5) and one outlet layer (#7). All these layers were stacked in vertical direction from the bottom layer #7 to the top layer #1, successively. The channel layers (#1, #3 and #5) have a three-sheet structure of enclosing the patterned channels on the polymer film with the inlet and outlet covers (see Figure S1). Instead, the adhesive layers #2 and #4 are single-layer tapes patterned with holes that connect channels in adjacent layers or with cut-through channels that move the fluids laterally. The adhesive layer #6 was designed with two pieces of lateral channels which are responsible for adjusting the resistances of flow paths. The main parallel channel layer #1 consists of multiplexing channels for passively achieving the cell concentration in a high-throughput manner. In this work, the asymmetric serpentine channel was employed to focus cells into a train located at the channel centerline based on the principle of inertial focusing19,29. In serpentine channels, the centerline focusing of cells can be achieved in a continuous manner without the 3



assistance of the external fields or complex microstructures.3,30 For cells satisfying the criterion (𝑎𝑝 𝐻 ≥ 0.07, where 𝑎𝑝 is the particle diameter, and H is the channel height for low-aspect-ratio channels)31,32, the only requirement for achieving the inertial focusing is to drive the sample with a flow rate within a specific range. Moreover, the Dean flow induced by the channel curvature can assist in the speeding up of inertial focusing and thus reduce the required minimum channel length,3 which is benefit for reducing the device footprint. The massive parallelization of serpentine channels can be easily realized to increase the processing throughput. All these features make the serpentine channel be the ideal geometry for our MPIM device. In this work, a multiplexing structure of 12 radially-arrayed serpentine channels was designed for ultrahigh throughput cell concentration. For the current 12 radially-arrayed channel design, a throughput up to 8 ml/min can be easily provided, which we think is sufficient for processing most large-volume biofluids33 or environment samples34. The throughput of our MPIM device is identical to that of the reported 40 radially-arrayed straight channels (8 ml/min)26 and is even higher than that of the reported 256 parallel straight channels (2.5 ml/min)27. Further increasing the throughput of our MPIM device can be realized through stacking multiplex parallel channel layers. It is worth noting that in this work the flow rate is defined as the processing throughput because our MPIM device is commonly used for concentrating trace cells from a large background fluid.

Figure 1. (a) Explosion diagram of our MPIM device which was fabricated by vertically stacking multiplexing layers. (b) Design of the serpentine channel in the parallel channel layer (#1). (c) Capability of our MPIM device to move fluids in and across different layers. Figure 1(b) shows the structure of each serpentine channel. To further reduce the device footprint, we bended the serpentine channel to fully fill the area of a 30 degree sector. In doing so, 12 serpentine channels each with a channel length of over 60 mm 4


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can be radially arrayed in a 20 mm radius circle. Each repeated unit of the serpentine channel consists of a large curving turn with a radius of RL and a small curving turn with a radius of RS. The width of the small turn was designed to be constant at WS while the large turn smoothly connects with the small turn and has variable widths (the largest width of the large turn was designed to be WL). In this work, three types of serpentine channels (Types I, II and III, WS/H=3, 4 and 5) were designed and the detailed dimensions of each channel design are provided in Table S1. A simple guideline for designing the channel dimensions is provided in Supporting Information Section S-1. Figure 1(c) demonstrates the capability of our MPIM device to move fluids in and across different layers. After being injected into the device through the inlet, the samples will be equally distributed into 12 serpentine channels in the layer #1. Due to the inertial focusing effect in serpentine channels, the cells will be tightly focused and exported from the central outlet while the blank fluids will be exported through the two side outlets. The blank fluids will first pass through the vertical holes in the layer #2 and then flow into the layer #3. The layer #3 consists of 12 T-shaped channels for converging the blank fluids and the layer #4 laterally moves the converged blank fluids to the non-center region. Meanwhile, the concentrated samples will directly flow into the layer #5 through the vertical holes in layers #1~4. Similar to the T-shaped channels in layer #3, the radially-arrayed straight channels in layer #5 converge the concentrated samples towards the center region. Finally, the blank fluid and the concentrated sample will move laterally in the layer #6 and be respectively exported from the two outlets in the layer #7. Although multiplexing channels are parallelized, our MPIM device only has one inlet and two outlets, which is benefit for simplifying the operation procedure. Device fabrication and assembly The parallel and confluence channel layers (#1, #3 and #5) contain the enclosed channels and were fabricated in polymer films using the laser cutting and the roll-toroll lamination. The fabrication method is described in the Supporting Information Section S-2 and Figure S2. The double-sided tapes were employed as the adhesive layers (#2, #4 and #6) and patterned with through holes or channels using laser cutting. A single sheet of polyvinyl chloride (PVC) film patterned with two outlets was employed as the outlet layer #7. Figure S3 shows the photographs of the finished each layer.

Figure 2. Photographs of the fabricated MPIM device at the angles of (a) front view and (b) side view. (c) Cross-section of the serpentine channel. 5



Figure S4 illustrates the assembly process of our MPIM device after fabricating each layer. To assist in assembling, four locating holes were designed at the corners of each layer and a custom fixture were employed. We realized the quick assembly of our MPIM device within three main steps by respectively layering different channel layers and adhesive layers (Supporting Information Section S-3). Figures 2(a) and 2(b) are respectively the photographs of the fabricated MPIM device. As can be seen from these figures, our MPIM device appears like a thin card while the thickness of our MPIM device is only 1.4 mm. Figure 2(c) is the cross section of a serpentine channel formed by sealing the through grooves on a PVC film between two polyethylene terephthalate films with thermal sensitive ethylene-vinyl acetate copolymer (PET/EVA films). To realize the purpose of “inset and play”, a custom housing was employed as the chip-toworld interface (Figure S5). Although these are 13 film layers stacked, our MPIM device shows an acceptable optical transparency (Figure S6) and the particle migration can be clearly observed (Supplementary Video S1). The good transparency indicates that our MPIM device can be expanded as a potential pre-focusing unit for highthroughput optical flow cytometers. The materials for fabricating our MPIM device are all very cheap (less than $0.1 for each device). The combination of plastic sheet, tape, and stacking makes our MPIM device practical for the low-cost and disposable use in resource-limited environments. In addition, our MPIM device can bear a high pressure without leakage which is sufficient for inertial microfluidic applications and the using of rigid plastic materials prevents the channel channel from significant deformation under high pressures. In future, our multilayer stacking method can be expanded for fabricating other devices with 3D layouts for enhancing mixing or realizing the multiunit lab-on-a-chip integration. Preparation of particle/cell suspensions The polystyrene particle solutions (diameters of 7 μm, 10 μm and 15 μm, Thermal Scientific, Inc) were diluted with the phosphate-buffered saline (0.01M, Sigma) containing 0.5 wt% Tween 20 (Sigma-Aldrich) to form the uniformly-dispensed particle suspensions with specific concentrations. In the application section, the marine unicellular green microalgae of GY-H1 Platymonas helgolandica tsingtaoensis was purchased from the company of shanghai guangyu biological technology and cultured in the F/2+Si medium according to the provided protocol. After harvesting, the microalgae suspensions were diluted with the culture media to specific concentrations. To mimic the rare circulating tumor cells in blood, the human breast adenocarcinoma cancer cells (MCF-7 cells) were spiked into the blood sample at the amounts of hundreds to thousands per milliliter blood. The MCF-7 cells were cultured in highglucose Dulbecco’s modified Eagle’s medium (DMEM, Life technologies) supplemented with 10% fetal bovine serum (FBS, Life technologies) and 1% penicillinstreptomycin (Life technologies) using a carbon dioxide incubator (Forma 381, Thermo Scientific). Human whole blood was draw from a healthy consenting volunteer and collected via a vacutainer collection tube (BD Biosciences) containing anticoagulant 6


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K2EDTA. The size distributions and initial concentrations of the prepared samples were analyzed using a Countess II FL automated cell counter (Thermo Fisher Scientific). Experimental setup and data analysis The fabricated MPIM device was sandwiched between two transparent PMMA plates with their inlet and outlets being sealing using the O rings. Then, the packed device was clamped by two machined metal housings to serve as the chip-to-world interface (Figure S5). The inlet and the two outlets on the housings were respectively connected with the syringe and the centrifuge tubes using the Teflon tubings. The syringe pump (Legato 270, KD Scientific) was employed to provide the precise flow rates for achieving the particle focusing. The collected liquids were sampled under a microscope (IX 71, Olympus) and then their particle concentrations were counted using a Countess II FL automated cell counter (Thermo Fisher Scientific). The particle focusing dynamics in a single serpentine channel were visualized via an inverted fluorescence microscope (IX 71, Olympus) equipped with a camera (Exi Blue, Qimaging). The image frames illustrating the particle distributions were recorded under the bright-field mode using a short exposure time of 10 μs. To avoid the random error, the discrete image frames over a certain time period were overlaid using the ImageJ software (NIH) to create the composite images.

Results and Discussion Understanding the physics behind the device Before testing our MPIM device, we first explored the inertial focusing behaviors in a single serpentine channel to provide a better understanding on the physics of our device. In this experiment, three types of serpentine channels with different WS/H of 3, 4 and 5 were tested. The suspensions with different particle sizes of 7 μm, 10 μm and 15 μm were respectively injected into the serpentine channels at the flow rates ranging from 100 μl/min to 700 μl/min (with an interval of 100 μl/min). The particle dynamics near the channel outlet were captured and stacked to create the composite focusing maps illustrating the particle distributions over a time period. Figure 3(a) illustrates the focusing maps of the three tested particles in the serpentine channel with a WS/H=3. The particle focusing maps in other two large channels (WS/H=4 and 5) were provided in Figure S7. From these focusing maps, we found that the randomly-dispensed particles will first focus into two steams, then the two particle streams gradually shift towards each other with increasing flow rate and finally the single stream focusing is formed at the channel centerline.

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Figure 3. (a) Focusing maps of 7 μm, 10 μm and 15 μm particles in a single serpentine channel with a WS/H of 3 at different flow rates ranging from 100 μl/min to 700 μl/min. (b) Focusing status map of 7 μm, 10 μm and 15 μm particles in the three tested channels. The preferred flow regimes of TBF-c and SSF modes in which the particles are exported from the central outlet are framed in black. (c) The overlaid focusing images illustrating the particles being exported from the two side outlets (in the TSF mode) and the particle band being exported from the central outlet (in the TBF-c mode). To provide a clear picture of particle focusing in serpentine channels, we divided the observed focusing phenomena into four modes including incomplete focusing (IF), two stream focusing (TSF), transitional band focusing (TBF) and single stream focusing (SSF) and plotted the focusing status map in Figure 3(b). It is interesting to find that the SSF and TBF will occur at lower flow rate for larger particles due to the 8


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strong inertial lift force (𝐹𝐿~𝑎4𝑝)35. To realize the function of particle concentration, the focusing is necessary to effectively gather the particles and then remove the particle-free fluids. Under the TSF mode, only the particle-free fluids between the two particle streams can be removed through the central outlet (Figure 3(c)). Instead, under the SSF mode, the focused particle stream can be exported from the central outlet while two parts of fluids near the side walls can be effectively removed from the two side outlets, which is more benefit for increasing the concentration of target samples. For the TBF mode, the particles can also be exported from the central outlet when the particle bands are within a specific width (Figure 3(c), this condition was renamed as TBF-c mode). To achieve a better concentration effect, the SSF and TBF-c modes are preferred for our channel design. We then marked the flow regimes of TBF-c and SSF modes in the focusing status map in Figure 3(b). It is observed that the serpentine channel with a WS/H of 3 owns the widest flow regime of TBF-c and SSF modes for stably exporting the focused particle streams/bands from the central outlet. Therefore, this channel design was selected for the following concentration applications. In addition to the concentration application, our channel design can be expanded for the separation of two differently-sized particles when these two particles are respectively in SSF and TSF modes. Characterization of concentration performances We next systematically characterized the concentration performances of our MPIM device. The 7 μm, 10 μm and 15 μm particle suspensions were pumped into our MPIM device at the flow rates of 1~8 ml/min (~12 times the flow rate tested in single channel) with an interval of 1 ml/min. For each particle suspension, the volumes and particle concentrations of the initial samples and the collected samples at different flow rates were respectively measured. To quantitatively evaluate the concentration performances of our MPIM device, two dimensionless parameters, the recovery efficiency (RE = 𝑛𝑡𝑎𝑟𝑔𝑒𝑡 𝑛 𝑐 𝑡𝑜𝑡𝑎𝑙) and the concentration factor (CF = 𝑡𝑎𝑟𝑔𝑒𝑡 𝑐𝑖𝑛𝑖𝑡𝑖𝑎𝑙), were defined. Here, 𝑛 is the particle number in the sample and is the product of particle concentration (𝑐) and sample volume (𝑣). Figure 4(a, b) illustrates the calculated RE and CF of these three particles at different flow rates. It is found that the 15 μm particles show relatively stable concentration performances over the whole flow rate range as the particles are in the preferred TBF-c and SSF modes (Figure 3(b)). The RE and CF of 10 μm particles will sharply increase and become similar to that of 15 μm particles after the flow rate is increased to be higher than 3 ml/min as the SSF mode is achieved after this flow rate (Figure 3(b)). For these two particles, a RE of over 95% (sometimes approaching 100%) and a CF approaching the theoretical value of 2.5 (this theoretical value was calculated using the flow splitting ratio (f) in Figure S8) can be achieved under most flow rates. When the flow rate was increased to the highest tested value of 8 ml/min, a slight deterioration of concentration performances was observed possibly due to the slight shifting of particle stream away from the channel centerline as observed in Figure 3(a). Instead, the concentration performances of 7 μm particles will always be improved with 9



increasing flow rate. A RE of 86 ± 4 % and a CF of 1.9 ± 0.1 can be achieved under the flow rate of 8 ml/min.

Figure 4. Concentration performances of our MPIM device. (a) RE and (b) CF of 7 μm, 10 μm and 15 μm particles at different flow rates of 1~8 ml/min. (c) RE and (d) CF of 10 μm and 15 μm particles under the initial concentrations of 104, 105 and 106 counts/ml. We then explored the effect of initial concentration on concentration performance. The flow rate was respectively fixed at 5 ml/min for 10 μm particles and 4 ml/min for 15 μm particles. Figure 4(c, d) illustrates the RE and CF of 10 μm and 15 μm particles under three initial concentrations of 104, 105 and 106 counts/ml. It is found that at the initial concentrations of 104 and 105 counts/ml, our MPIM device exhibits an expected ideal performance with the RE of 93~99% and the CF of over 2.3 for all these two particles. Further increasing the concentration to 106 counts/ml, the performance will deteriorate due to the enhanced particle-particle interactions. The ability to process the samples with initial concentrations up to 106 counts/ml makes our MPIM device capable of processing various practical samples. In addition, our MPIM device is especially suitable for concentrating trace cells from a large-volume background fluid which is difficult to be achieved using the traditional centrifugation method7. Applications We next applied our MPIM device for concentrating biological cells to showcase its utility. In the first application, high-throughput microalgae harvesting from a largevolume media was demonstrated. The marine unicellular green microalgae of GY-H1 Platymonas helgolandica tsingtaoensis (Figure S9(a)) with a non-spherical flat shape and polydisperse sizes of 5~20 μm (an average of 12 μm) was employed. The initial 10


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concentrations of microalgae suspensions were controlled to be at the level of 105 counts/ml. Figure 5(a, b) shows the RE and CF at different flow rates of 1~8 ml/min. It is found that the concentration performances will first gradually improve with increasing flow rate and then slightly deteriorate at high flow rates, which agrees well with the above observation of 10 μm particles. Under the optimal flow rate of 6 ml/min, a RE of 90 ± 2 % and a CF of 2.3 ± 0.2 can be achieved.

Figure 5. Concentration performances of microalgae. (a) RE and (b) CF at different flow rates of 1~8 ml/min. (c) RE and CF after performing each serial concentration step (C1~C4). (d) An example showing the concentration variation of target samples during a 4-step serial concentration. To achieve a higher CF for practical applications, a multi-step serial concentration was performed through reinjecting the collected target samples into our MPIM device. The flow rate was controlled to be at the optimal value of 6 ml/min for each concentration step. Figure 5(c) illustrates the RE and CF of each concentration step (C1~C4). It is found that the concentration performances keep stable during the first three concentration steps (C1~C3). In the C4 step, the concentration performance slightly deteriorates as the cell concentration approaches 106 counts/ml. Figure 5(d) and Figure S9(b) show an example of the concentration changes during a 4-step serial concentration. Final concentration of microalgae is increased by 16.1 times after performing the 4-step serial concentration. Meanwhile, with gradually increasing the cell concentration, the volume of the target sample reduces from 50 ml to 2~3 ml after the C4 step. Next, we employed our MPIM device as an “on-chip centrifuge” for highthroughput concentration of trace tumor cells from a large-volume background fluid. The isolation and detection of rare circulating tumor cells (CTCs) from human whole blood serve as a new “liquid biopsy” technique for effectively monitoring the cancer 11



metastasis at its early stage.36 However, after being separated from the large background blood cells, the CTCs commonly remain to be suspended in a large-volume fluid and thus an additional volume reduction step is still highly needed before performing the downstream biochemical analysis. In the current work, we employed our MPIM device for high-throughput concentrating the separated tumor cell sample to a small volume. We first employed the pure MCF-7 cell samples with initial concentrations of 105 count/ml to test the concentration performances of our MPIM device. Figure 6(a) shows the RE and CF at different flow rates of 1~8 ml/min. A “first increase and then decrease” variation of RE and CF was observed, which is similar to that observed in the above experiments using particles or microalgae. However, the overall concentration performances for MCF-7 tumor cells are significantly better than those for microalgae due to the better focusing effect of larger tumor cells. Under the optimal flow rate of 5 ml/min, a RE of 94 ± 2 % and a CF of 2.3 ± 0.2 can be achieved. The averages of RE and CF over the whole flow rate range are respectively ~90% and 2.2.

Figure 6. Concentration performances of tumor cells. (a) RE and (b) CF at different flow rates of 1~8 ml/min. (c) The workflow of our MPIM device for concentrating the separated trace tumor cells. The microscopic images illustrate the concentrations of the initial sample (after separation) and the final sample (after concentration). After searching for the optimal flow rate, we further applied our MPIM device for concentrating the separated tumor cells. First, the whole blood sample spiked with trace tumor cells was processed using our previously-reported microfluidic instrument37 (Step 1 in Figure 6(c)). The rare tumor cells can be separated from the large background blood cells at a high recovery ratio of ~85% within a rapid processing time of 15 min. However, the volume of the separated sample has reached over 15 ml. Then, a multi12


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step serial concentration was performed to concentrate the trace tumor cells from the obtained 15 ml sample using our MPIM device (Step 2 in Figure 6(c)). After concentration, the original 15 ml sample has been successfully reduced to only ~0.7 ml. Figure 6(c) shows the microscopic images of the initial and final samples. The unstrained background white blood cells were excluded using the fluorescence observation mode. It is found that the concentration of tumor cells has significantly increased after the multi-step serial concentration. Through counting the cell number in the sampled windows (N>3), the CF was estimated to be around 14 and a high RE of over 80% was achieved even when processing such low concentration samples. Further increasing the RE can be realized by recycling the cells in the waste samples using a closed-loop recycling system. The Trypan blue exclusion test indicates that a cell viability of over 96% is kept after processing by our MPIM device. We envision the wide application of our MPIM device as a potential low-cost “on-chip centrifuge” for environment analysis and disease diagnosis.

Conclusions In this work, a novel MPIM device with complex 3D fluidic paths was fabricated through stacking the enclosed channel layers and the patterned adhesive layers. Our MPIM device offers the new fluid handling capability that are difficult to achieve using conventional PDMS devices. As a prototype demonstration, a MPIM device consisting of 12 radially-arrayed serpentine channels but having only one inlet and two outlets was fabricated for achieving the high throughput cell concentration. Then, the effects of flow rate, particle size and initial concentration on concentration performance were systematically explored. The experimental results show that our MPIM device is capable of concentrating various particles at a throughput up to 8 ml/min. Under the optimal flow rate, a RE approaching 100% and a CF of 2.5 can be easily achieved. Finally, we successfully applied our MPIM device for the concentration of the microalgae and trace tumor cells from large background fluids. To achieve a high CF, the multi-step serial concentration is performed. We envision the wide application of our MPIM device as a low-cost “on-chip centrifuge” for the pretreatment of various bioparticles in the areas of environment analysis and disease diagnosis.

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: Three-sheet structure and fabrication method of the channel layers, photographs of the fabricated each layer, assembly process of the MPIM device, chip-to-world housings for clamping the MPIM device, photograph of the MPIM device, focusing maps of 7 μm, 10 μm and 15 μm particles in serpentine channels with WS/H of 4 and 5, flow splitting ratios of the MPIM device, size distribution of the green microalgae and the microscopic images of the samples during a 4-step serial concentration, dimensions and design guideline of the serpentine channels, descriptions on the process for fabricating 13



and assembling the MPIM device, and the video illustrating the particle migration in the MPIM device.

Conflicts of interest There are no conflicts to declare.

Acknowledgements This research work is supported by the National Natural Science Foundation of China (81727801, 51875103, 51775111 and 51505082), the Fundamental Research Funds for the Central Universities (2242017K41031), the Six Talent Peaks Project of Jiangsu Province (SWYY-005) and the ZhiShan Young Scholar Fellowship.

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A multilayer polymer-film inertial microfluidic device for high

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