Inertial microfluidic syringe cell concentrator - Analytical Chemistry

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Inertial microfluidic syringe cell concentrator Nan Xiang, Xin Shi, Yu Han, Zhiguo Shi, Fengtao Jiang, and Zhonghua Ni Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02201 • Publication Date (Web): 12 Jul 2018 Downloaded from http://pubs.acs.org on July 14, 2018

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Analytical Chemistry

Inertial microfluidic syringe cell concentrator Nan Xianga*, Xin Shib, Yu Hana, Zhiguo Shia, Fengtao Jianga, and Zhonghua Nia* a 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] b Department of General Surgery, Zhongda Hospital, Southeast University, Nanjing, 210009, China.

Abstract Low-cost, easy-to-use cell concentration tools are in urgent demand for biomedical diagnosis in resource-poor settings. Herein, we propose a novel inertial microfluidic syringe cell (IMSC) concentrator that employs inertial focusing to increase cell concentration through ordering the cell and removing the cell-free fluid. A three-part structure, consisting of a cap-shaped upper housing, a circular gasket and a lower housing with a spiral channel, is adopted for simple fabricating and assembling, which enables the seamless translation of our IMSC concentrator into commercial outcomes without additional redesigning. The performance characterization indicates that our IMSC concentrator is capable of processing samples with different initial concentrations over a broad flow rate range. The satisfactory concentration performances over a broad driving flow rate range make it possible for our IMSC concentrator to be driven by pushing the syringe with single hand. Finally, pollen particles and MCF-7 cells are successfully concentrated at a high throughput of 3.0 ml/min (up to 4.2×107 counts/ml) under the hand-powered drive. We envision wide applications of our IMSC concentrator as “centrifugation on a syringe tip” to various cell concentration pretreatments in resource-poor settings.

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Introduction Concentrating biological cells to a desirable concentration is a crucial sample pretreatment step frequently performed in biochemical or clinical laboratories. For example, the concentration of rare pathogens or parasites in body fluids (e.g., blood, urine and pleural effusion) may enable the early diagnosis of disease to be possible.1-3 Currently, centrifugation is still the most widely employed sample concentration technique which involves the application of centrifugal force induced by high-speed rotations to settle target cells from solution and the removal of supernatant with a pipette.4 Although the centrifugation has been regarded as the “gold standard” for concentrating micro/nanomaterials, it may not suitable for use in resource-poor settings as the commercial centrifuge is bulky and requires electricity-powered. In addition, the centrifugation is incapable for concentrating samples with small volumes or with low concentrations due to the cell loss under a heavy centrifugal effect.5-7 Another classic scheme for cell concentration is microfiltration, which employs micropores of specific sizes to capture the cells and remove the cell-free fluid.8 However, it is difficult to release the captured cells and avoid the clogging issue. To address the above issue, researchers have made continuous efforts to develop affordable centrifugation techniques that can be used in resource-poor settings. Up to now, various wonderful ideas inspiring from our daily life have been proposed for low-cost centrifugation. For example, some research groups adopted the kitchen utensils (e.g., egg beater9 and salad spinner10) as low-cost centrifuges for isolating the plasma from whole blood. Recently, the playful whirligig was also used as the ultralow-cost, lightweight and human-powered centrifuge, which could achieve a strong centrifugal force of 30,000 g.11,12 Although the ideas are very fantastic and interesting, it is difficult to promote the use of these do-it-yourself (DIY) centrifuges for researchers from different disciplines. The advent of microfluidics has provided new insights for cell concentration. As the offered special advantages (such as low sample consumption, acceptable hardware cost and easy to miniaturize), the microfluidic concentration technique has attracted increasing interests in recent years.13,14 According to the working principles, previous works on microfluidic concentration can be divided into two categories. The one category is the technique of “centrifugation on a chip” which evolves from the concept of traditional centrifuge-based concentration.15,16 Instead of using a centrifuge tube, a microfluidic compact disc (CD) was applied and spun by a motor system like a CD driver to generate the centrifugal force required for settling cells. Recently, the toy of spinning top, which transforms the hand-powered pull-out operation into the rotation motion, was proposed to drive the microfluidic CD.17 However, for technologies of this category, the samples can only be processed in a batch mode and the generated centrifugal force is not strong enough for completely settling a very small number of cells in the solution. Another category can be named as “concentration on a chip” which utilizes the forces induced by external fields or hydrodynamic effects to concentrate cells in microfluidic channels. The active field-based schemes achieve the increase of particle 2


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concentration via attracting the around cells towards specific locations under the external fields (electrical18-20, magnetic21,22, acoustic23,24, etc.). However, the recovery of the gathered cells and the using of bulky external field generators are still the major problems preventing the active schemes from application in resource-poor settings. The passive hydrodynamic schemes achieve cell concentration through focusing the cells into a narrow stream/band and then removing the blank fluid without cells.25 As compared with active ones, the passive schemes own the advantages of low hardware requirement, high processing throughput, simple structure and easy to operate, and are the ideal choice for concentration applications in resource-poor settings. In this field, a lot of excellent works using inertial microfluidics6,26-29 or hydrophoresis30,31 for continuous cell concentration are springing up. However, nearly all the reported microfluidic concentrators are clean room products fabricated in polydimethylsiloxane (PDMS) using the complicated soft lithography and the designs of concentrators are still difficult to directly translate to commercial outcomes. Herein, we propose a novel inertial microfluidic syringe cell (IMSC) concentrator for high-throughput and continuous cell concentration. The key functional module of our IMSC concentrator is the spiral inertial microfluidics which can focus the cells into a train without external field forces and thus achieves the cell concentration via removing the cell-free fluid. A design of three-part structure including a cap-shaped upper housing, a circular gasket and a lower housing with a spiral channel is proposed, which enables the seamless translation of our IMSC concentrator into commercial outcomes without additional redesigning step. After fabricating the prototype device, the concentration performances at different driving flow rates and initial concentrations are systematically characterized. Finally, the pollen particles and MCF-7 cells with complex features are concentrated under the drive of single-handedly pushing syringe.

Conceptual design Figure 1 illustrates the conceptual design of our novel IMSC concentrator, which can achieve the high-throughput and continuous cell concentration through simply injecting samples at specific flow rates. Our IMSC concentrator appears as a Φ34 mm disk-shaped housing with one inlet at the center of one surface and two outlets at another surface. The inlet was designed to be in standard female luer lock format, which enables an easy and leak-free connection with various commercially available syringes for quick sample injection. The designed male slip luer outlets can be connected with luer stub adapters or other fittings for exporting the concentrated cell samples and the blank liquid. The positioning of two outlets with a spacing of 9 mm fits well with the well distance in microtiter plates (e.g., 96-well plates) so that the concentrated cell samples can be directly dispensed into the microwells and applied for further culture and biological analysis. In our IMSC concentrator, a spiral inertial microfluidic channel with a low aspect-ratio (AR=channel height (H)/channel width (W)) of 0.2 was employed. As previously-explored in our and others’ studies32-35, the 3



cells flowing in spiral channels could be focused into cell trains at finite Reynolds numbers. This interesting cell self-focusing phenomenon, named as inertial focusing, can be explained as the force balance between the inertial lift force (FL) caused by the shear gradient and the Dean drag force (FD) caused by the cross-sectional secondary flow.36-38 Both of these two forces have their directions perpendicular to the main flow. A more detailed mechanisms on inertial focusing can be found in many previous studies.39-41 In this conceptual design, we applied the inertial focusing for cell concentration through adopting a proper outlet system to extract the focused cell trains and remove the blank fluid. As the whole focusing and extracting processes rely only on the inherent inertial effects of flowing fluid, our IMSC concentrator does not need any bulk and expensive external field generators (e.g., electrode, magnet and interdigital transducer) or complex microstructures (e.g., micropore and micropillar). The only requirement for our IMSC concentrator is to provide the sample with a driving flow rate in a certain range. To lower the power for driving the sample at a high throughput up to ml/min level, we designed a 3-loop spiral geometry channel with a large cross-sectional dimension of 100 µm (H)×500 µm (W) for our IMSC concentrator. Other channel dimensions are provided in Table S1.

Figure 1. Conceptual design of our novel IMSC concentrator which employs the inertial focusing in spiral channels for high-throughput and continuous cell concentration. To verify our conceptual design, we first experimentally explored the focusing performances in a traditional PDMS spiral channel which is identical to that in our IMSC concentrator. The PDMS spiral channel was replicated using the soft lithography technique.42 Four polymer particle solutions with different diameters of 10 µm, 20 µm, 25 µm and 30 µm were pumped into the fabricated spiral channel at the flow rates of 1.5~4.0 ml/min (with an interval of 0.5 ml/min). The Reynolds numbers were calculated to be 83~222 under the tested flow conditions. All these four particles satisfy the inertial focusing criterion, ap/H≧0.07, where the ap is the particle diameter.36,43 The particle distributions near the outlet are provided in Figure 2 to 4


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clearly illustrate the effect of flow rate on particle focusing. From this figure, it is found that particles gradually focus into a single train with increasing flow rate and then dispense into multi-trains at high flow rates, agreeing well with previous observations.34,35 More importantly, all the 10 µm and 20 µm particles are observed to have migrated into the inner half channel at all tested flow rates while the large 25 µm and 30 µm particles occupy their lateral positions in inner half channel at the flow rates above 2.0 ml/min. On the basis of this observation, a Y-shaped outlet with two equally-bifurcated branches was employed to reduce the particle loss and provide an effective driving flow rate range of 2.0~4.0 ml/min. The feature of such broad driving flow rate range makes our IMSC concentrator can be simply driven through manually pushing syringes or through other portable fluid driving tools (e.g., fluidic pipette or dispenser). For the current device, the particles with diameters ranging from 7 µm to 60 µm can be successfully focused according to the previous theory.36,44 To focus the particles smaller than 7 µm, a spiral channel with a smaller cross section can be designed.

Figure 2. Focusing performances of differently-sized particles (10 µm, 20 µm, 25 µm and 30 µm) in a PDMS spiral channel at the flow rates of 1.5~4.0 ml/min (with an interval of 0.5 ml/min). The top walls in these bright-field microscopic images are the inner walls.

Device fabrication and assembly As shown in Figure 3(a), our IMSC concentrator consists of three parts, including a cap-shaped upper housing, a circular gasket and a lower housing with a spiral channel. The CAD drawings clearly illustrating the detailed structures of each part are 5



provided in Figure S1(a). The three parts can be easily assembled by using the screw threads which are respectively designed on the sides of upper and lower housings. After screwing home the housings, the semi-open spiral channel on the top surface of the lower housing will be tightly sealed with the silicone gasket, as illustrate in Figure 3(b). The large spacing of 3.25 mm between the adjacent channel loops also ensures a leak-free sealing of the spiral channel under a strong stress. To enhance the sealing, a thin layer of medical epoxy resin adhesive was spun onto the surface of gasket. After being partially cured, the gasket can be adhered to channel surface without affecting the channel structures. It is worth noting that the luer lock inlet on the upper housing, the inlet orifice in the gasket and the inlet reservoir of the spiral channel are all positioned at the center. This design makes sure that the screwing of lower housing does not affect the alignment of these three inlets.

Figure 3. (a) Structure diagram of our IMSC concentrator which contains three parts: a cap-shaped upper housing, a circular gasket and a lower housing. (b) The cutaway view of the assembled concentrator illustrating the fit between the three parts. (c) Photograph of the assembled concentrator of which the upper and lower housings are fabricated via 3D printing. (d, e) The planar and cross-sectional profiles of the spiral channel on the lower housing. Figure 3(c) shows the photograph of the assembled IMSC concentrator. As a proof-of-concept demonstration, here the two housings are respectively fabricated in photocurable resins using the stereolithography (SLA) three dimensional (3D) printing. Instead of creating the whole device directly using 3D printing, the three-part design enables the seamless translation of our IMSC concentrator into commercial outcomes without additional redesigning step. In addition, the large channel dimensions (e.g., the cross-section of 100 µm×500 µm) employed indicates that our IMSC concentrator can be manufactured like other disposable medical devices by using the well-established injection moulding in the traditional plastic industry. We believe that the cost of our IMSC concentrator will be lower than that of the classic syringe filter due to the simple structure. Figure 3(d, e) illustrates the microscopic images of channel planar and cross-sectional profiles, respectively. To replicate the cross-sectional profile, the 3D printed housing was casted with mixed PDMS liquid 6


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and cut perpendicular to the channel after being curing. From these two figures, it is found that the 3D printed channel shows an acceptable surface quality and a good verticality of side walls. Besides 3D printing, we also fabricated our IMSC concentrator prototype in metal aluminum using the traditional mechanical milling technique. The photographs of the fabricated concentrator and its microscopic images illustrating the planar and cross-sectional structures with good qualities are provided in Figure S1(b-d). For the metal concentrator, it is possible to reuse the concentrator via a thorough cleaning after unscrewing the housings. To further reduce the fabrication cost, we will separate the key channel structure from the housings as a disposable unit using our previously-explored low-cost polymer film chip technique.32,45

Usage method We describe two usage methods for operating our IMSC concentrator to achieve the cell concentration, as illustrated in Figure 4. In the first protocol, a pump is employed to drive the sample and an intravenous flow regulator is placed between the syringe and the IMSC concentrator for simply controlling the driving flow rate (see Figure 4(a)). In this work, we employed this relatively accurate flow driving method for characterizing the performance indexes of our IMSC concentrator. In addition to the syringe pump used in Figure 4(a), various pumps (e.g., peristaltic pump, medical infusion pump and so on) available in laboratories and clinics can be adopted. Through integrating the flow regulator into the fluidic circuit, even the low cost industry pumps with large output flow rates (Qmin > 4 ml/min) can be used.

Figure 4. Usage methods for operating our IMSC concentrator to achieve the cell concentration. (a) Samples driven by the syringe pump for characterizing the performance indexes of our IMSC concentrator. (b) The output flow rates when rolling the wheel to different marked positions (from #1 to #10). The samples are driven by single-handedly pushing the syringe according to the provided instruction 7



[The injection of 10 ml samples is required to be completed within 120 ± 10 seconds at a uniform speed]. Each flow rate in this figure is the average of different operators’ data (N=5). (c) Samples directly driven by single-handedly pushing the syringe. The red ink is artificially added to sample just for purpose of illustrating the concentration process. For the practical applications, we envision that our IMSC concentrator can be operated without using bulk and power consuming pumps. As the driving flow rate range of our IMSC concentrator is relatively broad, we expect that the slight variation of driving flow rates will not significantly affect the concentration performances. Inspiring of this fact, we replaced the pump with manually pushing the syringe. Five operators with different body mass indexes were asked to single-handedly push the syringe fully-filled with 10 ml samples at a uniform speed. The injection of 10 ml samples is required to be completed within an acceptable operating time of 120 ± 10 seconds, which yields a driving flow rate of about 5 ml/min. Other specific flow rates smaller than 5 ml/min can also be easily obtained via rolling the wheel in the intravenous flow regulator. To monitor and measure the flow rates generated by single-handedly pushing the syringe, we built the experimental setup in Figure S2 to record the increase of fluid mass on the electronic balance in every 10 seconds. Figure 4(b) illustrates the output flow rates at different wheel positions (a scale was used to mark the wheel positions). It is found that the output flow rate decreases linearly when the wheel rolls from position #1 to position #8 due to the increase of flow resistance caused by the tube deformation. At positions #9 and #10, the tube is totally blocked so that the output flow rate is zero. For experienced operators, it is easy to directly operate the IMSC concentrator without using the flow regulator, as illustrated in Figure 4(c). In future, we will integrate the Quake-style microvalves46,47 into our concentrator to make the concentration performance totally independent of operation.

Characterization of performance indexes To characterize the performance indexes of our IMSC concentrator, monodispersed polymer particles with diameters of 10 µm and 20 µm were respectively injected into the concentrator inlet using the sample driving method illustrated in Figure 4(a). The samples exported from the inner and outer outlets were respectively collected and analyzed. Figure 5(a) illustrates the microscopic images of the initial samples and the collected samples from inner and outer outlets at a driving flow rate of 2.5 ml/min. It is clearly found that nearly all the particles are exported from the inner outlet and the concentrations of the samples from the inner outlet significantly increase as compared with those of initial samples. This qualitative result clearly validates the effectiveness of our IMSC concentrator. To explore the effect of flow rate on concentration performance, we varied the driving flow rates from 1.5 ml/min to 4.0 ml/min with an interval of 0.5 ml/min. The volumes and particle concentrations of initial and exported samples at each flow rate were measured. Two dimensionless parameters, recovery efficiency (RE=ninner/ntotal) 8


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and concentration factor (CF=cinner/cinitial), were defined to quantitatively evaluate the concentration performances. Here, n is the particle number in the sample and is the product of particle concentration (c) and sample volume (v). Figure 5(b, c) illustrates the variations of RE and CF as a function of flow rate (all the samples were sampled more than five times). It is interesting to find that for both two sized particles, the concentration performances will first enhance with increasing flow rate and then deteriorate at high flow rates, which agrees well with the above-explored particle focusing phenomena. In addition, the small particles show better concentration performances at low flow rates while the large particles exhibit better performances at high flow rates because a much stronger flow is required to ensure the complete migration of large particles into inner channel region.

Figure 5. (a) Microscopic images of the initial samples (inlet) and the collected samples from inner and outer outlets at a driving flow rate of 2.5 ml/min. The scale bar is 150 µm. (b) Recovery efficiency (RE) as a function of flow rate. (c) Concentration factor (CF) as a function of flow rate. The optimal flow rate of 3.0 ml/min for 20 µm large particles to achieve the best concentration performance (i.e., the highest values of RE and CF) is slightly higher than that for 10 µm particles (2.5 ml/min). Under the optimal flow rate, the RE of 10 µm particles is 100%, indicating that all the particles are successfully recovered. The corresponding CF of 10 µm particles after a single run approaches 2.20 which even slightly exceeds the theoretical CF of 2.00 using the Y-shaped outlet system. The 9



achieved optimal RE and CF of large 20 µm particles are respectively 92.47% and 1.96, which are slightly worse than those of 10 µm particles. In addition to the flow rate, the particle concentration would affect the particle inertial focusing, as demonstrated in previous studies.48,49 To explore the effect of particle concentration on concentration performance, the two-sized particle solutions with different initial concentrations of 104, 105 and 106 counts/ml were respectively injected into the concentrator inlet under the optimal flow rates. Figure 6(a, b) illustrates the performance indexes, REs and CFs, at different initial particle concentrations. At the initial concentrations of 104 and 105, our IMSC concentrator exhibits an expected ideal performance with RE approaching 100% and CF of about 2.00. Further increasing the initial concentration to 106 counts/ml, a slight deterioration of concentration performance could be observed due to the increased particle-particle interactions at high concentrations. The upper limit of particle concentration can further be increased to be more than 107 (data see below). This broad concentration range that can be processed makes our IMSC concentrator capable of processing various practical samples. As the special advantage of microfluidics lies in dealing with small volume samples with extremely low concentrations,6,7 our IMSC concentrator is especially suitable for concentrating rare cells (e.g., circulating tumor cells after being separated from blood). For rare cells, the cell recovery is especially important while an RE of 100% can be easily achieved using our IMSC concentrator.

Figure 6. (a) Recovery efficiency (RE) and (b) concentration factor (CF) at different 10


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initial particle concentrations of 104, 105 and 106 counts/ml. The driving flow rates for the 10 µm and 20 µm particles were controlled to be the optimal flow rates (i.e., 2.5 ml/min for 10 µm particles and 3.0 ml/min for 20 µm particles). (c) Microscopic images and (d) photographs of samples collected from inner outlet after performing the 3-step serial concentration (N1, N2 and N3). (e) Quantitative data illustrating the particle concentrations in the initial sample and the samples collected after each concentration step (N1, N2 and N3). In the N1 concentration step, the particles in the samples collected from outer outlet are not observed. The using of equally-bifurcated outlet limits the CF after a single run to about 2. To achieve a higher CF for extremely dilute samples, a serial concentration can be performed via reinjecting the collected target samples into our IMSC concentrator. Figure 6(c-e) illustrates an example of 3-step serial concentration (N1, N2 and N3) for the 10 µm particle solution with an initial concentration of 18.27 ± 0.49 ×104 counts/ml. As can be seen from the sampled microscopic images and photographs of collected samples in Figure 6(c, d), the particle concentrations are obviously increased and the colors of collected sample liquids gradually turn green after each step. Verifying by the quantitative data in Figure 6(e), a CF of 7.59 can be easily obtained after performing the 3-step serial concentration and the RE after N3 step is calculated to be over 96%. With gradually increasing the particle concentration, the volume of target sample has successfully reduced from 13 ml to only 1.6 ml after the N3 concentration step. The whole operating procedure for this 3-step serial concentration can be completed within about 10 min, which is affordable for the hand-powered operation. Further increasing the CF can be realized through progressively extracting the cell-free fluid along the whole channel.6

Applications for concentrating bioparticles To explore the practicability of our IMSC concentrator for processing the real biological samples, pollen particles and MCF-7 cells were tested. The aim of this test is to verify whether our IMSC concentrator is capable of processing bioparticles with complex shapes, polydisperse sizes and deformable features. Specifically, the pollen particles have an acanthosphere shape and their sizes were measured to be in the range of 15~24 µm with a main peak around 20 µm. The MCF-7 cells are deformable spheres with diameters of 15~25 µm (an average of 20 µm). Before the single-handedly pushing applications, the concentration performances of these two bioparticles at varied flow rates were first characterized to optimize the driving flow rate. The initial particle concentrations of the tested samples were controlled to be in the level of ~105 counts/ml. Figure S3(a) illustrates the REs of these two bioparticles at varied flow rates of 1.5~4.0 ml/min. From this figure, it is found that the variation of RE with increasing flow rate is similar to that for 20 µm polymer particles. The optimal flow rates for these two bioparticles both are determined to be 3.0 ml/min, which agrees well with the above data of 20 µm polymer particles. Under the optimal flow rate, a RE of ~92% can be achieved for both bioparticles. In addition, it is found that the pollen particles exhibit better concentration performances than the MCF-7 11



cells although they have a similar size distribution. The reason for this difference can be explained as the additional deformation-induced force which shifts the focusing position towards the channel centerline and thus makes more cells be exported from the outer outlet. After searching for the optimal driving flow rate, we prepared three particle samples with different initial concentrations (i.e., pollen particles and MCF-7 cells at concentrations of ~105 counts/ml, and pollen particles at a very high concentration of 107). Then, the three prepared samples were respectively injected into the concentrator inlet using the protocol illustrated in Figure 4(c). According to the provided usage method, several operators were asked to single-handedly push the syringe to generate a relatively uniform driving flow rate of 3.0 ± 0.5 ml/min over the whole pushing period. The generated driving flow rates were monitored using the experimental setup in Figure S2. Figures 7(a) and S3(b) illustrate the photographs and microscopic images of initial samples and samples collected from outlet when processing the pollen particle samples with two different concentrations of 107 and 105 counts/ml. The processing throughput is up to 4.2×107 counts/ml. These results clearly show that the samples have been significantly concentrated after running through our IMSC concentrator. To give a clear picture of our IMSC concentrator’s concentration performances, average REs of these three bioparticle samples are illustrated in Figure 7(b). For pollen particle samples with a high concentration of 107, an average RE of about 72% can be achieved while for low concentration samples, the average RE can reach 90%. For deformable MCF-7 cells with a concentration of 105 counts/ml, the average RE is over 85%. Under the drive of single-handedly pushing the syringe, the concentration performances are slightly worse than the data in Figure S3(a), which is possible due to the flow fluctuating during the pushing process.

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Figure 7. (a) Photographs and microscopic images of initial pollen particle samples and samples collected from inner and outer outlets. The initial particle concentration researches 107 counts/ml in this experiment. The scale bar in the microscopic images is 200 µm. (b) Average REs of the three tested bioparticle samples (pollen particles with cinitial= 107 counts/ml and 105 counts/ml; MCF-7 cells with cinitial= 105 counts/ml) driven by single-handedly pushing the syringe. Each data is the average of REs from different operators (N=5). For biological cells, the shear stress in high-speed inertial flows may induce the damage of the flowing cells. A trypan blue exclusion test was performed to evaluate the cell viability of the collected samples after running through our IMSC concentrator. It is found that a cell viability of over 96% is kept even for samples collected at the highest driving flow rate of 4.0 ml/min. Further validating the safety of our IMSC concentrator, the collected cells were re-cultured for over 96 hours. Figure S4 is the microscopic images of cultured cells at different time scales, which indicates a negligible shear-stress effect on cell viability. In addition, previous study also proves the negligible shear-stress effect on cell from the aspect of the gene expression profile before and after flow.50 In addition to the above tested cell/bioparticle samples, our IMSC concentrator could be applied for concentrating various biological cells in large-volume biofluids (e.g., the concentration of trace blood cells in urine samples for the early diagnosis of hematuria).

Conclusion In this work, a novel IMSC concentrator, which consists of three parts including a cap-shaped upper housing, a circular gasket and a lower housing with a spiral channel, was proposed for high-throughput and continuous cell concentration. The concentrator can be easily assembled by screwing the two housings and the three-part design enables the seamless translation of our IMSC concentrator into commercial outcomes without additional redesigning. Then, the effects of flow rate and particle initial concentration on concentration performances were characterized. It is validated that our IMSC concentrator is capable of concentrating samples with particle concentrations up to 107 counts/ml at flow rates of 2.0~4.0 ml/min. The acceptable concentration performances over a broad driving flow rate range make our IMSC concentrator be driven by single-handedly pushing the syringe. Through performing a 3-step serial concentration, a CF of 7.59 and a RE of over 96% can be easily obtained. Finally, the pollen particles and MCF-7 cells were employed to verify the practicability of our IMSC concentrator. Under the drive of single-handedly pushing the syringe, an average RE of about 72% can be achieved for pollen particle samples with a high concentration of 107 counts/ml. For the pollen particle and deformable MCF-7 cell samples with low concentrations of 105 counts/ml, the average REs can respectively reach over 90% and 85% without affecting the viability of cells. We 13



envision that our IMSC concentrator can be widely employed as “centrifugation on a syringe tip” tool for sample pretreatment in resource-poor settings.

Experimental section Experimental setup for characterizing the particle focusing The particle focusing performances in the conceptual design section was characterized using the transparent PDMS device which was fabricated using the well-established soft lithography and replica molding techniques.42 Specifically, a master mold was fabricated through selectively patterning the layer of 100 µm thick SU-8 photoresist (SU-8 2150, Microchem) on a silicon wafer. Then, the degassed polydimethylsiloxane (PDMS) liquid (Sylgard 184, Dow Corning) with the ratio of base to curing agent of 10:1 was poured onto the master mold. After curing at 80 °C for 3 h in an oven, the PDMS block with spiral channels was peeled from the master mold and cut into small pieces. The chip-to-world orifices were carefully punched via a stub adapter. Finally, the prepared PDMS pieces were irreversibly bonded with the glass sides using an oxygen plasma cleaner (PDC-002, Harrick Plasma). The particle focusing dynamics in the spiral channel were visualized using an inverted fluorescence microscope (IX 71, Olympus) equipped with a CCD camera (Exi Blue, Qimaging). The particle samples were pumped into the channel using the syringe pump (Legato 270, KD Scientific). The videos illustrating the particle migrations were recorded under bright-filed mode using an exposure time of 10 µs. To clearly illustrate the particle focusing and avoid the random error, the discrete video frames over a certain time period were overlaid using the ImageJ software (NIH) to create the composite images. Experimental setup for characterizing the IMSC concentrator The upper and lower housings used in our IMSC concentrator were fabricated using a laser based stereolithography (SLA) 3D printing system (3DSL-450S, DigitalManu) in high-resolution photocurable resin (SZUV-W8001). To ensure the printing quality of microchannel structures, the lower housing was printed from the outlet plane to the spiral channel plane. The selected material has been proven to be non-toxic and is widely employed for printing various biomedical devices. In future, our IMSC concentrator can be directly manufactured in medical plastics. The samples in the IMSC concentrator were driven by the syringe pump (Legato 270, KD Scientific) or by handedly pushing the syringe. An intravenous flow regulator (ZY-1, tube diameter of 3.5 mm, Zhiyu Medical Instrument) was employed to adjust the flow rate. The concentration performances of the IMSC concentrator were characterized by analyzing the collected samples. The particle concentrations were observed under a microscope and then counted using a Countess® II FL Automated Cell Counter (Thermo Fisher Scientific).

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Particle and cell sample preparation The polystyrene standard-sized particle solutions (diameters of 10 µm, 20 µm, 25 µm and 30 µm) with an initial solid content of 1% were purchased from Thermal Scientific, Inc. Human breast adenocarcinoma cancer cells, MCF-7 cells, were grown to confluence in high-glucose Dulbecco’s modified Eagle’s medium (DMEM, Life technologies) supplemented with 10% fetal bovine serum (FBS, Life technologies) and 1% penicillin-streptomycin (Life technologies) in a carbon dioxide incubator (Forma 381, Thermo Scientific). After dissociating using Trypsin-EDTA solution (Life technologies), MCF-7 cells were re-dispersed in sterile phosphate-buffered saline (0.01M, Sigma). The collected cells were re-cultured using the above protocol and periodically inspected using the microscope. The pollen particles (Ragweed pollen, cat No. 214) were purchased from Thermal Scientific, Inc. All the employed particles and cells were mixed into the phosphate-buffered saline (0.01M, Sigma) containing 0.5 wt% Tween 20 (Sigma-Aldrich) to form homogeneous suspensions with controlled particle/cell concentrations.

Supporting Information CAD drawings of our IMSC concentrator, photographs and microscopic images of the fabricated aluminum concentrator, experimental setup for monitoring and measuring the flow rate, Res and microscopic images of pollen particles and MCF-7 cells at varied flow rates, microscopic images of cells cultured for different time scales, and the detailed dimensions of the designed spiral channel.

Corresponding Authors Nan Xiang: *E-mail: [email protected]. Phone: +86-025-52090518. Fax: +86-025-52090501. Zhonghua Ni: *E-mail: [email protected]. ORCID Nan Xiang: 0000-0001-9803-4783 Acknowledgements This research work is supported by the National Natural Science Foundation of China (51505082, 81727801, 81572906 and 51775111), the Natural Science Foundation of Jiangsu Province (BK20150606), the Fundamental Research Funds for the Central Universities (2242017K41031) and the Six Talent Peaks Project of Jiangsu Province (SWYY-005). References (1) Mach, A. J.; Adeyiga, O. B.; Di Carlo, D. Lab on a Chip 2013, 13, 1011-1026. 15



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Inertial microfluidic syringe cell concentrator - Analytical Chemistry

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