Elasto-inertial microfluidic separation of microspheres with submicron resolution at high-throughput (1).pdf
Jeon et al. Microsystems & Nanoengineering   (2024) 10:15  Microsystems & Nanoengineering https://doi.org/10.1038/s41378-023-00633-w www.nature.com/micronano A R T I C L E O p e n A c c e s s Elasto-inertial microﬂuidic separation of microspheres with submicron resolution at high-throughput Hyunwoo Jeon1, Song Ha Lee1, Jongho Shin2, Kicheol Song2, Nari Ahn2 and Jinsoo Park1✉ Abstract Elasto-inertial microﬂuidic separation offers many advantages including high throughput and separation resolution. Even though the separation efﬁciency highly depends on precise control of the ﬂow conditions, no concrete guidelines have been reported yet in elasto-inertial microﬂuidics. Here, we propose a dimensionless analysis for precise estimation of the microsphere behaviors across the interface of Newtonian and viscoelastic ﬂuids. Reynolds number, modiﬁed Weissenberg number, and modiﬁed elastic number are used to investigate the balance between inertial and elastic lift forces. Based on the ﬁndings, we introduce a new dimensionless number deﬁned as the width of the Newtonian ﬂuid stream divided by microsphere diameter. The proposed dimensionless analysis allows us to predict whether the microspheres migrate across the co-ﬂow interface. The theoretical estimation is found to be in good agreement with the experimental results using 2.1- and 3.2-μm-diameter polystyrene microspheres in a co-ﬂow of water and polyethylene oxide solution. Based on the theoretical estimation, we also realize submicron separation of the microspheres with 2.1 and 2.5 μm in diameter at high throughput, high purity (>95%), and high recovery rate (>97%). The applicability of the proposed method was validated by separation of platelets from similar-sized Escherichia coli (E.coli). Introduction Sample preparation is an essential step in the overall chemical analysis process; it improves analytical results by enriching the target material or removing contaminants before analysis1. Particle manipulation techniques have received increasing research attention owing to their applications in sample ﬁltration2, ﬂow cytometry3, and separation4. Various microﬂuidic manipulation techni- ques with high throughput, resolution, and efﬁciency have been developed. In particular, regarding resolution, techniques for the precise sample separation such as red blood cells (7–8 μm)5, platelets (1.5–3 μm)6, and bacteria (1–3 μm)7 are highly required. However, the submicron- resolution separation of micro-objects is difﬁcult because most of the forces moving the particles in the lateral direction are proportional to the sample size8. Polymer microspheres have been widely utilized as model objects for bio-particles9,10. Passive manipulation techniques rely on internal hydrodynamic forces acting on suspended objects, whereas in active techniques, forces are applied to the objects from external actuation devices. The typical examples of the passive techniques include inertial ﬂow focusing based on ﬂuid inertial force11,12, pinched ﬂow13 and Dean ﬂow fractionation based on Dean drag force generated in curved pipes14,15, and deterministic lateral displacement (DLD) based on the streamline of ﬂow along the structures16. These techni- ques allow simple device conﬁguration and manipulation, offer high single-chip throughput, and enable paralleli- zation. However, numerous parameters such as the microchannel geometry, ﬂuid properties, and ﬂow con- ditions need to be strictly tuned; otherwise, the accuracy of sample separation, efﬁciency, and throughput may be © The Author(s) 2024 OpenAccessThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,whichpermitsuse,sharing,adaptation,distributionandreproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Correspondence: Jinsoo Park (jinsoopark@jnu.ac.kr) 1Department of Mechanical Engineering, Chonnam National University, 77 Yongbong-ro Buk-gu, Gwangju 61186, Republic of Korea 2Analytical Engineering Team, Samsung Display Co., Ltd., 181 Samsung-ro, Tangjeong-myeon, Asan-si, Chungcheongnam-do 31454, Republic of Korea 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; compromised. Conversely, in active techniques, opto- phoresis17, dielectrophoresis18, magnetophoresis19, and acoustophoresis20 are applied to manipulate the target samples in an on-demand manner. Despite high precision, the device conﬁguration and parallelization are complex, and high-cost and sophisticated equipment are usually required compared to the passive approaches. Elasto-inertial microﬂuidics using viscoelastic ﬂuids can offer a breakthrough by providing increased sample manipulation performance in addition to the advantages of conventional inertial microﬂuidics. In a viscoelastic ﬂuid ﬂow, the difference in the non-uniform normal stress acting on the suspended objects generates an elastic lift force (Fel), an additional force that enables the lateral migration of the object21–23. Micro-objects, mostly on microspheres, manipulation studies using viscoelastic ﬂuids24–26 have achieved a single equilibrium position of the microspheres9,27, in contrast with conventional iner- tial microﬂuidics, where particles have more than two equilibrium positions28. Moreover, elasto-inertial micro- ﬂuidic separation using a co-ﬂow of Newtonian and vis- coelastic ﬂuids have been recently conducted to improve the sample separation performance29–34. The co-ﬂow allows the wall-induced lift force (Fw) and shear-gradient lift force (Fs), which drive the lateral displacement of the particles suspended in the Newtonian ﬂuid, to promote or suppress the migration of the particles owing to the generation of Fel; this force can be used to increase the separation resolution. To identify the correlation between these forces under various ﬂow conditions, dimensionless numbers can be applied. In general, in the co-ﬂow of Newtonian and viscoelastic ﬂuids, Reynolds number (Re)29, Weissenberg number (Wi)35, and elastic number (El = Wi/Re)29,36 have been previously applied for theo- retical analysis. However, from the viewpoint of particle behaviors, we found that these dimensionless numbers have limitations, especially in the co-ﬂow conﬁguration of elasto-inertial microﬂuidics. In this study, we introduced modiﬁed Wi (Wim) and modiﬁed El (Elm = Wim/Re) for investigation of the microsphere lateral migration across the interface of Newtonian and viscoelastic ﬂuids. For thorough valida- tion, we conduct a series of experiments with polystyrene (PS) particles with diameters of 2.1 and 3.2 μm under varying ﬂow conditions, in which the proposed modiﬁed El is found to provide a better understanding of the sus- pended object behavior in the co-ﬂow elasto-inertial microﬂuidics. We further introduce a new dimensionless number deﬁned as the Newtonian ﬂuid stream width divided by the microsphere diameter. Based on this dimensionless number, we determine three regimes of inertial focusing, elasto-inertial transition, and elastic focusing for the lateral migration of microspheres from Newtonian to viscoelastic ﬂuids across the interface. Based on theoretical ﬁndings, we could precisely predict the microsphere behavior and thus achieve the submicron separation of the PS microspheres with diameters of 2.1 and 2.5 μm, as well as 2.5 and 3.2 μm at high throughput, high purity, and high recovery rate. The proposed elasto- inertial microﬂuidic separation was applied for separation of similar-sized bio-particles: platelets and Escherichia coli (E. Coli) to validate the practical applicability. Working mechanism Device conﬁguration and elasto-inertial separation of microspheres Figure 1a schematizes the microﬂuidic device, com- posed of two inlet ports and three outlet ports, for the elasto-inertial separation of microspheres. A sample ﬂuid (DI water solution with suspended PS microspheres) and a sheath ﬂuid (dilute polyethylene oxide (PEO) solution) were introduced through inlets 1 and 2, respectively. The sample ﬂuid ﬂow, represented in sky blue in Fig. 1, was bifurcated at the upstream to sandwich the sheath ﬂuid in the center, presented in orange, at midstream to form a three-layered co-ﬂow of the sample/sheath/sample ﬂuids. Figure 1b represents the sequential transfer of the microspheres along the microchannel in the regions i–iv marked in Fig. 1a. The microspheres suspended in the Newtonian ﬂuid are initially aligned along the two side- walls of the microchannel by the viscoelastic sheath ﬂuid ﬂow (Fig. 1b (i)). The microspheres near the walls experience a wall-induced lift force (Fw) owing to the increased pressure between the wall and the microspheres and consequently migrate away from the wall toward the Newtonian/viscoelastic ﬂuid interface. Because Fw is proportional to the microsphere diameter (d) such that Fw ∝d6, the larger microspheres experience Fw with greater magnitude and thus migrate faster away from the wall compared with the smaller microspheres (Fig. 1b (ii))37. The larger microspheres positioned across the New- tonian/viscoelastic ﬂuid interface begin to experience an elastic lift force (Fel) induced by the elastic effect of the viscoelastic ﬂuid. The elastic lift force can be expressed as Fel = Celd3∇N1, where Cel is the elastic lift coefﬁcient, N1 = σxx – σyy is the ﬁrst normal stress difference, and σxx and σyy are the stress tensor of the normal and transverse directions in the ﬂuid ﬂow, respectively. The ﬁrst normal stress difference can be expressed as N1 = 2μpλγ̇2, where μp is the polymeric contribution to the solution viscosity, λ is the relaxation time, and γ̇ is the average ﬂuid shear rate obtained using the Oldroyd-B constitutive model38. Fel acts toward the side wall when the majority of the microspheres are located in the Newtonian ﬂuid (Fig. 1b (ii)), whereas it acts toward the microchannel center when the majority of the microspheres are located in the vis- coelastic ﬂuid (Fig. 1b (iii))33. The smaller microspheres approach the Newtonian/viscoelastic ﬂuid interface after Jeon et al. Microsystems & Nanoengineering  (2024) 10:15  Page 2 of 14 precise prediction of the microsphere migration phe- nomenon across a co-ﬂow of Newtonian and viscoelastic ﬂuids. The proposed analysis is based on Reynolds num- ber, modiﬁed Weissenberg number, modiﬁed elastic number, and newly introduced dimensionless number deﬁned as the Newtonian ﬂuid width divided by the microsphere diameter. Using these dimensionless num- bers, we categorize the elasto-inertial microsphere lateral migration phenomenon into three regimes of inertial focusing, elasto-inertial transition, and elastic focusing based on particle equilibrium positions with reference to a co-ﬂow interface of Newtonian and viscoelastic ﬂuids. We experimentally validated the proposed analysis method with 2.1 and 3.2 μm PS microspheres under various ﬂow conditions. We found that our estimation method can better characterize the microsphere lateral migration in elasto-inertial microﬂuidics. In addition to high-efﬁciency (>99%) separation of 2.1 and 3.2 μm microspheres, we further achieved the submicron-resolution separation of 2.1 and 2.5 μm, as well as 2.5 and 3.2 μm, PS microspheres at high throughput, purity, and recovery rate. For valida- tion of practical applicability, we applied the proposed method for separation of similar-sized bio-particles: pla- telets from E. coli. We believe that the proposed dimen- sionless analysis can provide guidelines for the successful working conditions and estimation prior to experiments in the ﬁeld of elasto-inertial microﬂuidic sample separa- tion and puriﬁcation. Experimental section Device fabrication A master mold to fabricate Polydimethylsiloxane (PDMS) microchannels was prepared by a photo- lithography process using a negative photoresist E. coli Platelet Purity Recovery rate E. coli Platelet Performance (%) Minor axis (m) Major axis (m) E. coli 10.0kV 10.1mm×5.00k SE(UL) 10.0kV 10.2mm×5.00k SE(UL) 10.0um 10.0um Platelet 5 4 3 2 1 0 100 75 50 25 0 0 1 2 3 4 5 6 e d b a c Side outlet Center outlet Fluorescent  image Fluorescent  image Filtered  image Filtered  image E. coli only Platelet only Fig. 7 Escherichia coli and Platelet separaion. a Scanning electron microscope images of E. coli on the left side and platelet on the right side. b The length of major and minor axis of E. coli (black circle) and platelet (red circle). c The trajectories of E. coli and platelet under volumetric ﬂow rate condition of Qn = 10 μL min−1 and Qve = 50 μL min−1. E. coli and platelet move toward the side outlet and center outlet, respectively. Scale bar = 100 μm. d The efﬁciencies of E. coli and platelet separation in terms of purity and recovery rate. e The hemocytometer images of E. coli and platelet collected from the side outlet and center outlet. Scale bar = 50 μm Jeon et al. Microsystems & Nanoengineering  (2024) 10:15  Page 12 of 14 (SU-2050, Kayaku, japan). The microchannel with a rec- tangular cross-section was fabricated by a soft lithography replica molding process. The PDMS base and curing agent (Sylard 184 A and 184B, Dow Corning, USA) were mixed in a ratio of 10:1 (w/w%), poured into the master mold, and left in an oven at 80 °C for 2 h. The PDMS stamp, in which the microchannel was patterned, was bonded with a slide glass by oxygen plasma bonding (Covance, Femto Science, Korea). The microﬂuidic chip was placed in an oven at 65 °C for 2 h to further enhance the bonding strength. The main-microchannel was fabricated with a uniform height of 50 μm. The midstream microchannel, where the Newtonian and viscoelastic ﬂuid co-ﬂow is formed, was designed with a width of 20 μm. The channel length for the particles to have the equilibrium position in a rec- tangular channel was calculated by Lf = πμh2/ρUmd2Cli— where Um is the maximum ﬂow velocity37—to be 11.87 mm at the slowest ﬂow condition (Qtotal = 2 μL min−1) and the smallest particle diameter (d = 2.1 μm) used in this experiment. The length of the midstream microchannel L was designed to be 15 mm so that all particles have the equilibrium position in the channel. In this study, we approximated that each particle has an equilibrium position under all ﬂow conditions. Sample preparation Two types of viscoelastic ﬂuid with a concentration of 100 ppm were prepared by mixing PEO (Mw = 600 kDa, Sigma Aldrich, USA) powder in DI water for microsphere separation and 1× phosphate buffered saline (PBS) for bio-particle separation. The PEO solution was used after mixing with a magnetic stirrer for more than 24 h to ensure the complete dissolution of the PEO powder. Relaxation time of the 100 ppm PEO solution was obtained by the empirical formula λ = 18λZ(c/c*)0.65 (ref. 43). Overlapping concentration is expressed as c* = 0.77/[μ], and Zimm relaxation time λZ = F[μ]Mwμs/ NAkBT—where F is the pre-factor 0.463, μs is the solution viscosity, NA is Avogadro’s number, and kB is Boltzmann’s constant—is determined according to Zimm’s theory44,45. The theoretical values of c* and λZ can be obtained using the intrinsic viscosity [μ] = 0.072Mw 0.65 for the PEO solution according to the Mark–Houwink relationship46. The λ value of the 100-ppm PEO solution was calculated as 0.123 ms. We used the theoretical viscosity value of 100-ppm PEO solution for universal application of dimensionless analysis. The viscosity of PEO solution was calculated by 1.041 mPa∙s using polymer solution viscosity formula μ = μs + μp where μs is the solvent viscosity and μp = [μ]cμs is the polymeric contribution to the viscosity. The density of the synthesized PEO solution was mea- sured using a density meter (DMA 35 Basic, Anton Paar, Austria). The microsphere sample ﬂuid was prepared by mixing PS particles with DI water. For particle trajectory analysis, the sample ﬂuids were the mixture of d = 2.1 μm (green ﬂuorescent particle, Thermo Fisher, USA) and 3.2 μm (red ﬂuorescent particle, Thermo Fisher, USA) particles. Each sample used for submicron-resolution particle separation was mixed with d = 2.1 vs 2.5 μm (non-ﬂuorescent par- ticle, Thermo Fisher, USA) and 2.5 vs 3.2 μm particles. In all sample ﬂuids, the concentration of each sized particle was 1 × 107 particles mL−1, and the ﬁnal particle con- centration of the sample ﬂuid was 2 × 107 particles mL−1. For all microsphere sample ﬂuids, Tween 20 (Sigma Aldrich, USA) was mixed at a concentration of 0.1 v/v% to prevent particle aggregation. Optionally, PEO coating inside the microchannel47 and device manufacture with copolymers PDMS-polyethylene glycol48 could be used to prevent adsorption of particles to the channel walls, but these methods were not used in this study due to the sufﬁcient Re. The platelets were provided from the Kor- ean Red Cross and stored in shaker-incubator (ES-20, Grant bio, UK) at 22 °C. Before the experiments, the platelets were dyed with red ﬂuorescence by using an antibody labeling kit (Alexa FluorTM 568, Invitrogen, USA) and diluted 20 times with 1× PBS. The E. coli sample was cultured in a sterilized Luria-Bertani (LB) broth (L2542, Sigma Aldrich, USA) on a shaker-incubator at 37 °C for 24 h and diluted 10 times with 1× PBS. Flow visualization and measurements The Newtonian/viscoelastic ﬂuid interface was visua- lized by dispersing d = 300 nm (red ﬂuorescent, Thermo Fisher, USA) PS particles in DI water at a concentration of 1 × 108 particles mL−1. PS particles with d = 300 nm were injected in the direction of inlet 1 using a syringe pump (neMESYS Cetoni GmbH, Germany). Under the experi- mental conditions of this study, nanoparticles cannot migrate in the direction of the viscoelastic ﬂuid owing to the Fel caused by the PEO solution. In addition, the Newtonian/viscoelastic ﬂuid interface was measured at 100 μm near the upstream of the midstream micro- channel. Therefore, the diffusion effect was not con- sidered. Visualized images were precisely captured with a resolution of 0.0562 μm pixel−1 using an inverted microscope (IX73, Olympus, Japan), a CCD camera (E3ISPM, RisingCam, Japan). Microsphere trajectories were observed at the down- stream of the microchannel using an inverted microscope and captured via a high-speed camera (VEO 710 L, Phantom, USA). Images of the submicron-resolution separation were obtained by simultaneously using a halogen lamp and a mercury lamp, with ﬂuorescent par- ticles (2.1 and 3.2 μm) showing white color and non- ﬂuorescent mono particles (2.5 μm) showing black color (Fig. 6a). Jeon et al. Microsystems & Nanoengineering  (2024) 10:15  Page 13 of 14 Acknowledgements This work was supported in part by Samsung Display Company Ltd. and in part by the National Research Foundation of Korea (NRF) grants funded by the Korean government (MSIT) (Nos. RS-2023-00210891 and 2020R1A5A8018367). Author contributions H.J. and J.P. initiated and conceived the proof-of-concept experiments; H.J. designed and fabricated the microchannel; H.J., J.S., K.S., N.A., and J.P. characterized the devices and analyzed the results; H.J. and S.L. performed bio- particle experiments; H.J. and J.P. wrote the manuscript. All authors reviewed the manuscript. Competing interests The authors declare no competing interests. Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s41378-023-00633-w. Received: 30 May 2023 Revised: 23 October 2023 Accepted: 8 November 2023 References 1. Hyötyläinen, T. Critical evaluation of sample pretreatment techniques. Anal. Bioanal. Chem. 394, 743–758 (2009). 2. Broyles, B. S., Jacobson, S. C. & Ramsey, J. M. Sample ﬁltration, concentration, and separation integrated on microﬂuidic devices. Anal. Chem. 75, 2761–2767 (2003). 3. Olsen, L. R., Leipold, M. D., Pedersen, C. B. & Maecker, H. T. 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Title: Elasto-inertial microfluidic separation of microspheres with submicron resolution at high-throughput ​
Authors: Hyunwoo Jeon, SongHa Lee, Jongho Shin, Kicheol Song, Nari Ahn, Jinsoo Park ​
Publisher: Microsystems & Nanoengineering, Nature Publishing Group ​
Date: 22 January 2024​
Abstract: Elasto-inertial microfluidic separation offers many advantages including high throughput and separation resolution. ​ Even though the separation efficiency highly depends on precise control of the flow conditions, no concrete guidelines have been reported yet in elasto-inertial microfluidics. ​ Here, we propose a dimensionless analysis for precise estimation of the microsphere behaviors across the interface of Newtonian and viscoelastic fluids. ​ Reynolds number, modified Weissenberg number, and modified elastic number are used to investigate the balance between inertial and elastic lift forces. ​ Based on the findings, we introduce a new dimensionless number defined as the width of the Newtonian fluid stream divided by microsphere diameter. ​ The proposed dimensionless analysis allows us to predict whether the microspheres migrate across the co-flow interface. ​ The theoretical estimation is found to be in good agreement with the experimental results using 2.1-and 3.2-μm-diameter polystyrene microspheres in a co-flow of water and polyethylene oxide solution. ​ Based on the theoretical estimation, we also realize submicron separation of the microspheres with 2.1 and 2.5 μm in diameter at high throughput, high purity (>95%), and high recovery rate (>97%). ​ The applicability of the proposed method was validated by separation of platelets from similar-sized Escherichia coli (E. coli).