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Magnetic beads separation characteristics of a microfluidic bioseparation chip based on magnetophoresis with lattice-distributed soft magnets
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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Research articles
Magnetic beads separation characteristics of a microfluidic bioseparation chip based on magnetophoresis with lattice-distributed soft magnets Yunfeng Zhu, Biao Zhang, Jialiu Gu, Songjing Li
T
⁎
Department of Fluid Control and Automation, Harbin Institute of Technology, Harbin 150001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microfluidics Magnetic bead Bioseparation chip Soft magnet Magnetic field Separation
To continuously perform pretreatment operations of biological detection reagents, a kind of microfluidic bioseparation chip that integrates mixer, heater and soft magnets for detection of nucleic acid and extraction of molecular proteins with magnetic beads methods is presented in the paper. A specific magnetic field is formed in a desired chamber bonded with PDMS and PMMA sheets by embedding soft magnets of iron powders in the chip, and magnetized by an external magnetic field. Soft magnets distributed in different ways are designed respectively, and the characteristics of the magnetic field, as well as the magnetic trajectory of the magnetic beads are simulated by considering the magnetic-fluid coupling. An experimental platform to observe the capturing of magnetic beads is built and the capture phenomenon for magnetic beads with lattice-distributed soft magnets is also experimentally verified. Based on the experimental data, the capture efficiency of the magnetic beads is calculated with the image processing method. The results show that lattice-distributed soft magnets are more effective than the array-distributed soft magnets for separating the magnetic beads. Furthermore, the latticedistributed soft magnets can improve the separation efficiency of the detected reagent maintained to be more than 80% when the inlet flow rate increases from 0.01 m/s to 0.05 m/s.
1. Introduction Sample pretreatment plays an important role in biomedical analysis for early diagnosis of diseases, detection of nucleic acid and extraction of molecular proteins. Routine phenol-chloroform method is a typical way of DNA extraction because it provides high-quality templates for amplification, but the step of proliferation in this method is makes this method time-consuming. The membrane adsorption method uses a silica gel membrane to absorb DNA with a high salt buffer solution and elutes DNA with a low salt TE buffer solution after washing several times. However, the disadvantage of easy pollution limited the application of membrane adsorption method. As a timesaving method of high purity, magnetic bead method became more and more widely used in nucleic acid extraction [1–4]. Considering that only few particles in the sample are paramagnetic, the immune magnetic beads made by the core-shell synthesis method with superparamagnetic material and polymer composite material covered were used as carriers for transport in reagent [5,6]. Covalently attached to magnetic beads by the functional groups such as amino group, carboxyl group, and thiol group, analyte particles were manipulated to separate, detect and purify genes, proteins, cells and microorganisms by applying an external magnetic field [7–10]. Usually, the processes of extracting test samples involve ⁎
reagent mixing, cracking, heating and eluting with reagents placed in centrifuge tubes and transferred to different equipments. To reduce the cost of using large inspection equipments, functional devices were more recently fabricated in microfluidic chips, which were an effective technique for miniaturization and integration [11]. In the process of sample pretreatment, magnetic bead separation has always been the focus of research as the crux in the extraction steps since the efficiency of separation directly affects the amount of extracted substance. For particles or cells in a microfluidic device to move magnetically, the magnetic field and its gradient are the main influencing factors. Permanent magnet (NdFeB) was placed near the microchannel to generate the magnetic field and field gradient for simple installation and mass manufacturing. Improvement of processing and manufacturing technology makes it possible to integrate high gradient microscale permanent magnets in microfluidic chips [12,13]. Preliminary experiment integrated two micro-permanent magnets on two sides of the microchannel and separately recorded the separation of the magnetic beads when the permanent magnets were installed with the same pole and opposite poles [14]. Optimization studies were conducted on the separation characteristics of the magnetic beads with circular permanent magnets distributed outside the circular flow channel [15,16]. Similar experiments conducted on separation of
Corresponding author. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.jmmm.2020.166485 Received 19 October 2019; Received in revised form 18 January 2020; Accepted 18 January 2020 Available online 21 January 2020 0304-8853/ © 2020 Elsevier B.V. All rights reserved.
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
Y. Zhu, et al.
Fig. 1. Structure of microfluidic bioseparation chip.
a microfluidic bioseparation chip with miniaturized and integrated devices with lattice-distributed soft magnets is proposed that implements the mixture of samples, heating of reagents, and separation of magnetic beads, to accomplish the whole process of detection continuously instead of multiple manual operations. Since the number of separated magnetic beads determines the number of protein molecules and nucleic acids available for detection, it is meaningful to explore an appropriate structure of soft magnets to enhance the separation efficiency of magnetic beads in this chip.
magnetic beads under the action of a non-micro permanent magnet integrated microfluidic chip [17]. In these experiments, magnetic particles or cells were driven by stored magnetic energy of permanent magnets without any external power, but attenuation and uncontrollability of magnetic field inevitably affect the flexibility of the microfluidic devices. In the other hand, magnetic forces of magnetic beads with an integrated micro permanent magnets were not sufficient for high flow rates, and the non-cancellable magnetic field affects the recovery of the separated magnetic beads. Electromagnets were also used to generate magnetic fields for the flexibility in changing the strength of the magnetic field by controlling the current. The initial study produced electromagnetic fields for magnetic bead control by integrating wires at a microchannel [18,19]. Subsequent researches were mainly aimed at the joule heating effect when an electric current passes through the integrated electromagnet by integrated cooling channel on silicon-based magnetron microfluidic chip or placing the micro-coil outside the chip [20]. However, whether changing the material and shape of the wire or integrating the cooling device on the chip significantly increases the difficulty and cost of the microfluidic chip fabrication, and cannot eliminate the influence of the thermal effect on the detection solution. In recent research, soft ferromagnetic was implanted in a microstructure to produce a high gradient and non-uniform magnetic field with external magnetic field. The scientific community paid more attention to the microfluidic bioseparation technology with an integrated soft magnet structure due to its advantages, such as larger magnetic field force range, excellent controllability and wider applicability. Several experiments have integrated soft magnets on the sides of the microchannels to produce greater magnetic field strength, but the distributions of magnetic fields are relatively complex [21,22]. In recent research, micro soft magnets were distributed on the sides of a T-shaped flow channel and a L-shaped flow channel [23]. The materials of soft magnets were investigated to increase the magnetic field force in the channel. However, there are few studies in the influence of soft magnet arrangement around microchannels on magnetic field forces. Since the microfluidic bioseparation chip was proposed, it has been the focus of researches to integrate soft magnet or micro magnet on the chips. At present, fabrication of micro magnet is still following the microfluidic chip technology to pattern the shapes combining photolithography with etching techniques in silicon chip or glass and obtain the metal layers by sputtering, electroplating, evaporation and vapor deposition [24–28]. According to the material used in the micro magnet, iron powder or NdFeB, soft magnet or permanent magnet can be formed after the external magnetic field is magnetized [29,30]. In the existing researches, scientists mainly focused on the movement of magnetic beads to simulate cells manipulation in blood vessels. However, during the whole process of biological test, magnetic beads not only pass through the rectangular microchannel but also are mixed and separated in several chambers with different volumes. In this paper,
2. Structure and working principle of the microfluidic bioseparation chip 2.1. Structure and fabrication of the chip In the pretreatment process of nucleic acid detection, the lysate is firstly mixed with the cells. After a nucleic acid is successfully lysed, it is adsorbed on a magnetic bead with the best effect at a specific temperature. After that, the magnetic beads with nucleic acids are separated in a specific chamber and the remaining solution is drained. The nucleic acids can be collected when the elution solution is mixed with the magnetic beads in the final. Therefore, the bioseparation chip is designed including the mixer, heater and separation chamber. The above process would be repeated several times for some different nucleic acids, so it is necessary to add another flow channels and chambers for mixing and heating after the separation chamber. In this condition, the efficiency of magnetic beads separation determines the number of magnetic beads in the subsequent processing and greatly influences the entire nucleic acids extraction and subsequent detection results. As shown in Fig. 1, the microfluidic bioseparation chip consists of two layers respectively made by Polydimethylsiloxane (PDMS) and Poly Methyl Methacrylatemethacrylic Acid (PMMA). The PDMS layer is mainly used for mixture, reaction, and separation of the magnetic bead solution. It is formed with two fluidic inlets and one waste outlet on the other side of the layer, connected with three microfluidic channels and two chambers. Two channels extending from the inlets intersect and form a curved channel which is connected to the heating chamber with a larger height than the channels. In contrast, the separation chamber is manufactured between the heating chamber and the waste outlet with the same height of the channels. This microfluidic device is fabricated at the bottom of the PDMS layer by traditional soft etching technique. In particular, the mold of microfluidic channels and the separation chamber is manufactured by dry films and the heating chamber is shaped by a cylindrical mold. The PMMA layer includes the heating element, the soft magnets and a thin PDMS film covering the upper surface. Computer Numerical Control (CNC) is used to mill the grooves for the heating device and soft magnets, and than a piece of special metal is embedded into the grooves as the heating device. The heating foil is electroplated directly under the heating chamber and soft 2
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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linearly with the average magnetic field force of the magnetic beads at different positions in the chamber is used to describe the magnetic field force as
magnets are aligned with the separation chamber. Chosen as the material of soft magnets, iron powders are injected into the grooves milled by miniature engraving machine, and produce a gradient magnetic field in the separation chamber when they are magnetized by an external uniform magnetic field. A thin PDMS film is bonded to the PMMA layer by the surface treatment method, and it is bonded to the PDMS layer on other face.
1 Dh 0
P¯ =
h0
D
∫− D22 ∫− h2
0
→ → (H ·∇) H dxdz
2
The separation of the magnetic beads is mainly affected by the the magnetic field force component of in the vertical direction Fmz, which can be calculated by
2.2. Working principle Magnetic beads of micrometer or nanometer size are mixed with a dispersant to avoid agglomeration. The magnetic beads solution is injected into the microfluidic bioseparation chip and the sample reagent is poured into another inlet, keeping their quantities of flow in a constant ratio. Protein molecules or nucleic acids in the sample reagent are adequately mixed when flowing through the curved channel and adsorbed to functional groups on the surface of magnetic beads. The mixture from the curved channel is stored in the heating chamber which is designed to ensure the dose of test reagent and the biochemical reaction temperature through the heating foil under the chamber. After that, pulled by solution, the magnetic beads with the substance to be detected flow into the separation chamber to satisfy the requirement of subsequent eluent dosage. In this region, magnetic beads are affected by the magnetic field force of the soft magnets and change their directions of motion which were the same with the solution. Accordingly, the difference between two velocities causes fluid drag on magnetic beads to suppress the escape tendency of magnetic beads. When the magnetic beads are in micron scale, magnetic force, drag, gravity, and buoyancy are considered as the main forces in a magnetic-fluid coupling field, but the interaction between magnetic beads and Brownian force are ignored. The density of the magnetic beads used in this paper is similar to the solution, so the gravity and buoyancy of the magnetic beads can be neglected.
4μ0 πr 3χp
Fmz =
3 + χp
(Hx
4μ0 πr 3χp → ∂Hz ∂Hz ∂Hz + Hy + Hz )= (H ·∇Hz ) ∂x ∂y ∂z 3 + χp
(4)
It can be seen that the vertical component of the magnetic field force mainly depends on the magnetic field intensity H and the vertical component gradient of the magnetic field intensity ∇Hz . The permanent magnet that generates the external magnetic field is a cylindrical magnet with a radius of rm and a height of hm. If a coordinate system is created at the center of the bottom of the cylinder and (θ, zm) represents the point on the cylindrical surface, the components of the magnetic field intensity can be expressed as
Hx =
B0 dz m 4πμ0 μr
∫0 ∫0
2π
Hy =
B0 dz m 4πμ0 μr
∫0 ∫0
hm
2π
B0 dz m ( 4πμ0 μr
∫0 ∫0
hm
2π
hm
rm (z − z m) cos θ dθ K
(5)
rm (z − z m) sin θ dθ K
(6)
Hz =
−
hm
∫0 ∫0
2π
−rm (x − rm cos θ) cos θ dθ K
rm (y − rm sin θ) sin θ dθ ) K
K = [(x − rm cos θ)2 + (y − rm sin θ)2 + (z − z m)2]3/2 3. Calculation of magnetic force
(7) (8)
where θ is the angle between the x-axis and the perpendicular from the point to the z-axis; zm is the z coordinate of the point. The drag force coursed by velocity deviation of a magnetic bead Fd can be written as [33]
To study the trajectories of magnetic beads and the separation efficiencies of magnetic beads in the bioseparation chamber, the trajectory and magnetic field force of the magnetic beads in the above two soft magnets structures are respectively displayed by building the simulation models in a specific size. According to the simulation results, the separation efficiencies of magnetic beads in different magnetic fields are analyzed and compared.
→ Fd = 6πηr (→ vf − → vp) fD
(9)
where η and vf are the dynamic viscosity and velocity of the fluid respectively; fD is the hydrodynamic drag coefficient which is given by [34]
3.1. Effect of soft magnet
fD = [1 −
The magnetic force on a magnetic bead is given by [31]
4μ0 πr 3χp → → → Fm = (H ·∇) H 3 + χp
(3)
9 r 1 r 45 r 1 r ( )+ ( )3 − ( )4 − ( )5]−1 16 r + z ′ 8 r + z′ 256 r + z ′ 16 r + z ′ (10)
where z ′ is the distance between the magnetic bead and the surface of the microchannel. It can be seen that when the flow rate of the solution is higher or the magnetic bead are closer to the wall, the drag force of the magnetic bead Fd is larger. Then it will be more difficult to change the trajectory of this magnetic bead and capture it. As a result, the magnetic bead changes its trajectory due to these forces, either flowing to the drain channel with the solution or being adsorbed at the bottom of the chamber. The trajectory of the magnetic bead in the last movement fluctuation period before landing to the bottom is also displayed in Fig. 2. The direction of the magnetic field force points to the edge of the soft magnet at the beginning, with the magnetic bead moving down in the vertical direction. Then, the magnetic bead passes through the line 1 and the magnetic field force drags the magnetic bead upward until line 2 near the next soft magnet. After that, the magnetic bead is attracted to the second soft magnet again and finally captured at the edge where the magnetic field force is largest. In order to investigate the magnetic field force of the magnetic beads in the bioseparation chamber, several different magnetic field
(1)
where μ0 = 4π × 10−7 H/m is the magnetic permeability in the free space; r = 2.5 μm is the average diameter of the magnetic bead; χp = 0.2 is the magnetic volume susceptibility [32]; H is the magnetic field intensity at the center of magnetic bead. The following equation can be obtained from Eq. (1) as
→ → (H ·∇) H ∂Hy ∂Hy ∂Hy → → ∂H ∂H ∂H + Hz = i (Hx x + Hy x + Hz x ) + j (Hx + Hy ) ∂z ∂y ∂x ∂y ∂z ∂x → ∂Hz ∂Hz ∂Hz + k (Hx + Hy + Hz ) ∂x ∂y ∂z (2) It can be derived from Eqs. (1) and (2) that the magnetic force of the → → magnetic beads is linear with (H ∙∇) H when the material and size of the magnetic beads have been determined. A defined quantity that varies 3
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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field intensity are presented in Fig. 3b to d respectively. It can be seen from Fig. 3b that under the effect of a single permanent magnet with a magnetization of 500,000 A/m, the magnetic field intensity in the chamber is from |H| = 4.9 × 104 A/m to |H| = 5.1 × 104 A/m. When two identical permanent magnets are placed on both sides of the chamber in the same polarity direction, the magnetic field intensity in the chamber is almost constant and remains at |H| = 1.01 × 105 A/m. When the soft magnets are placed between the two permanent magnets, the magnetic field intensity above the soft magnets decreases dramatically with the distance to the soft magnets in the vertical direction but it increases with the distance to the chamber in the region between the soft magnets. The maximum magnetic field intensity |H| = 1.31 × 105 A/m is displaced at two edges of the soft magnet, and the minimum magnetic field intensity |H| = 7.89 × 104 A/m appears in the center of the gap. It is shown in Fig. 3c that the vertical component gradient model of the magnetic field intensity changes between |∇Hz| = 7.94 × 106 A/m2 and |∇Hz| = 8.26 × 106 A/m2 with a single permanent magnet. However, the vertical component gradient model of the magnetic field intensity increases significantly from |∇Hz| = 1.54 × 104 A/m2 in the center of the chamber to |∇Hz| = 6.02 × 105 A/m2 in both ends along the horizontal direction when two permanent magnets are placed. After integrating the soft magnets, the vertical component gradient mode of the average magnetic field intensity increases from |∇Hz| = 3.32 × 107 A/m2 to |∇Hz| = 8.26 × 106 A/m2, and the maximum value locates at the outer ends of the soft magnet. The magnetic field force is displayed in Fig. 3d where the vertical compo→ nent of magnetic field force (H ·∇) Hz increases from −3.99 × 1011 A2/ 3 11 2 3 m to −4.16 × 10 A /m with a single permanent magnet and it changes symmetrically in the range of −1.17 × 1011 A2/m3 to 1.17 × 1011 A2/m3 with two permanent magnets. After integrating the → soft magnets, variable of vertical magnetic field force (H ·∇) Hz changes 13 2 3 13 2 3 from −8.22 × 10 A /m to 2.23 × 10 A /m , with the distribution characteristics similar to the magnetic field intensity, and the direction of magnetic field force in the range of the soft magnet is opposite to that between the soft magnets. According to the above magnetic field distribution, the magnetic field intensity generated by a single permanent magnet is the smallest, but the vertical component gradient model of the magnetic field intensity is much larger than that of the two permanent magnets, causing that magnetic field force is much larger than the latter. For the same reason, as the soft magnets are integrated between the two permanent magnets, the magnitude of the magnetic field intensity is not changed much, but the vertical component gradient model of the magnetic field intensity can be increased by hundreds of times, thereby greatly increasing the magnetic field force in the chamber. Therefore, the soft magnets mainly increase the magnitude of the magnetic field force by increasing the gradient of the magnetic field intensity.
Fig. 2. Forces and motion trajectory of magnetic bead between two adjacent soft magnets.
Fig. 3. a) Side view schematic diagram of single permanent magnet, double permanent magnets and permanent magnets with soft magnets beside the separation chamber. b) Magnetic field intensity in the chamber. c) The vertical component gradient mode of the magnetic field intensity. d) Distribution of variable of magnetic field force in the vertical direction.
3.2. Magnetic force in bioseparation chamber To analyze the force and motion of a magnetic bead in the combination of magnetic field and flow field intuitively, a 2D simulation model is built to simulate the magnetic field force and fluid drag force on single magnetic bead in COMSOL and the trajectory of the magnetic bead is drawn. Ten magnetic stripes with widths of w = 0.2 mm, heights of h = 0.5 mm, and gaps of g = 0.3 mm are magnetized by the external uniform magnetic field with magnetic flux density of B = 0.1 T. The separation chamber has the diameter of D = 5 mm and height of h0 = 0.2 mm. The distance between the soft magnets and the bottom of the chamber is hf = 0.04 mm. As shown in Fig. 2, the background magnetic field is superimposed with the magnetic field of soft magnets excited by itself, and magnetic flux density is drawn as isopotential curves. The magnetic flux density within the length range of the soft magnet is larger than that between two soft magnets because
generation methods are compared. The magnetic field distributions in the separation chamber with a single permanent magnet, double permanent magnets and integrated soft magnets shown in Fig. 3a are simulated by the software COMSOL. Ten soft magnets of the same shape are generated, and each magnet has the width of w = 0.2 mm, the height of h = 0.5 mm and the gap of g = 0.3 mm. The height of PDMS film is hf = 0.04 mm. For the permanent magnet, the width is 10 mm, the height is 2 mm and the distance to the channel is 1.5 mm. The separation chamber has a diameter of D = 5 mm and a height of h0 = 0.2 mm. The contours for the magnetic field force and magnetic 4
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
Y. Zhu, et al.
Fig. 4. Schematic diagram of array-distributed soft magnets and lattice-distributed soft magnets.
mode of magnetic field intensity. It can be seen from the diagram that the gradient model of magnetic field intensity is the largest near the edge of a soft magnet and gradually decreases to both sides. There is a minimum of magnetic field intensity in the middle of a soft magnet and a smaller extremum between two adjacent magnetic stripes. In the whole separation chamber, the vertical component gradient mode of magnetic field intensity increases and decreases alternately along the flow direction. In particular, magnetic field intensity gradients of magnetic stripes on two sides of the soft magnets are remarkably larger than the others. As shown in Fig. 5b, the trajectory of the magnetic bead has a waving trend in the chamber, and the lowest points of each movement period appear at the back edges of the soft magnets. The magnetic beads generally move from the inlet to the outlet in the horizontal direction and present an up-and-down periodic motion in the vertical direction, eventually being captured near the edges of the soft magnets or escaping from the separation chamber. Along the direction from inlet to outlet in the chamber, the number of captured magnetic beads gradually reduces. When the magnetic beads entered the chamber, they are firstly subjected to the first magnetic stripe with the largest magnetic field intensity gradient. Therefore, the magnetic beads are mostly captured on the magnetic stripes in the front rows with a low inlet velocity.
the magnetic-field vector is in the same direction with the background magnetic field above the soft magnets and opposite in the gap. Obviously, the magnitudes and directions of the magnetic field forces only depend on the intensity of the magnetic field since the other physical parameters are constant. The direction of the magnetic field force is tangent to the direction of the magnetic field, that is, tangent to the magnetic induction line, and points from the low flux density to the high flux density. The magnetic field force vector distribution in the chamber is shown by the arrow in Fig. 2, where the magnetic field force is the largest at the edge and corner of the soft magnet, and gradually decreases at the surrounding space. 4. Design and calculation 4.1. Design of the lattice-distributed soft magnets The purpose of the design is to capture all the magnetic beads in the mixture at the bottom of the bioseparation chamber. In order to effectively enhance the magnetic field force and the intensity of magnetic field gradient, two distribution structures of soft magnets are displayed in Fig. 4. Array-distributed soft magnets consist of several stripe shape soft magnets fabricated from the inlet to the outlet of the bioseparation chamber, and the length of each soft magnet is the same with the diameter of the chamber. In the lattice-distributed structure, soft magnets are manufactured with multiple small cylinders and they are all confined within the bioseparation chamber. In particular, the number of columns of lattice-distributed soft magnets is always one more than the number of rows. The distance from the soft magnets to the bottom of the chamber is determined by the thickness of the PDMS film.
4.2.2. Distribution with lattice-distributed soft magnets According to the three dimensional simulation results in the last section, the gradient of magnetic field intensity is larger near the edges of the soft magnets. Thus, lattice-distributed soft magnets composed of small cylinders are implemented to increase the number of the soft magnet edges. Another three dimensional simulation model for soft magnets arranged at a matrix of 9 rows and 10 columns is built with the diameter of l = w = 0.2 mm, and gap of g = 0.3 mm. By keeping the remaining parameters unchanged, simulation results are drawn into diagrams of the trajectory of magnetic bead, magnetic field intensity gradient and distribution of captured magnetic beads in Fig. 6a and b. The average magnetic flux density mode in separation chamber is 0.11 T, which is similar to that of the array-distributed soft magnets, but vertical component gradient of magnetic field intensity is 1.527 × 108 A/m2, which is 18% larger than the former. Besides, the vertical component of the average magnetic field force is 1.115 × 10−12 N, 60% larger than that of the array-distributed soft magnets, whose average magnetic field force is 6.943 × 10−13 N. It can be seen from Fig. 6a that the gradient of the magnetic field intensity is larger near the edges of the soft magnets and the largest magnetic field intensity appears at the edge of every cylinder soft magnet, which is similar to array-distributed soft magnets. As shown in Fig. 6b, three magnetic beads are marked as examples for several typical trajectories in the separation chamber. The first magnetic bead passes through soft magnet directly after entering the chamber, and is captured rapidly at the edge of next soft magnet; the second magnetic bead has avoided the soft magnet at the beginning, and is still captured
4.2. Magnetic beads distribution 4.2.1. Distribution in array-distributed soft magnets A three dimensional model is also established to simulate distribution of the magnetic field in bioseparation chamber and trajectories of magnetic beads in this region. Parameters of simulation is the same with the 2D model and the length of magnetic stripe is l = 5 mm. Particle Tracing Module is chosen as the research method with three components which are Magnetic Fields No Currents (mfnc), Particle Tracing for Fluid Flow (fpt) and Laminar Flow (spf) respectively. The density and radius of particle are setting as 1000 kg/m3 and 2.5 μm, the relative permeability is 1000 and there are 300 particles ejected at the inlet. According to the results of numerical simulation, the average magnetic flux density mode in the bioseparation chamber increased by 0.01 T after the array-distributed soft magnets magnetized. However, the vertical component gradient model of the average magnetic field intensity increases from 0 to 1.297 × 108 A/m2, indicating that the soft magnets greatly enhance the gradient mode of the magnetic field intensity. Fig. 5a displays the distribution of vertical component gradient 5
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
Y. Zhu, et al.
Fig. 5. a) Distribution of vertical component gradient mode of magnetic field intensity in array-distributed soft magnets. b) Distribution of magnetic beads in arraydistributed soft magnets.
when it moves to the region of the soft magnet by the fluid drag force; the third magnetic bead is pulled straightly along the gap between the soft magnets where the vertical component of the magnetic force of magnetic bead is upward and the magnetic bead is finally captured onto the upper surface of the chamber. From the trajectories of the above three particles, it can be seen that the position of the magnetic field where the magnetic bead passes through in the chamber affects the trajectory of the magnetic bead. Moreover, when the magnetic bead closer to the channel wall, the velocity of magnetic bead is lower, and the drag force of the magnetic bead would be more important, reducing the velocity of magnetic bead at the entrance of the chamber and trapping the magnetic bead quicker. The final distribution of magnetic beads captured in the chamber is also displayed in Fig. 6b, showing that magnetic beads are captured more evenly with lattice-distributed soft magnets compared to the array-distributed soft magnets. The number of magnetic beads that are captured in the middle of the chamber increases but less magnetic beads are pulled to the outlet. Moreover, it is different from the array-distributed soft magnets that the separation of the magnetic beads can be effectively separated in the vertical direction in both the region of the soft magnets and the gap between the soft magnets. 5. Experiment and discussion In order to facilitate the observation of the magnetic beads distribution in the chamber after separation, the surfaces of the microfluidic chip are sealed with two films respectively to remain these two layers detachable. To record the captured magnetic beads, microfluidic bioseparation chip with array-distributed soft magnets and lattice-distributed soft magnets are fabricated in PMMA plates with the thickness of 1 mm, and covered by the thin PDMS films with the thickness of 0.02 mm. The PDMS layer with channels and chambers is also covered by another thin PDMS films with the thickness of 0.02 mm. The structure of the soft magnets can be observed through a microscope connected to the computer which is shown in Fig. 7. The array-distributed soft magnets are manufactured with the length of l = 5 mm, the width of w = 0.2 mm, height from h = 0.1 mm to 0.4 mm increasing by every 0.1 mm, and gap of g = 0.3 mm. The lattice-distributed soft magnets are manufactured with the radius from Rc = 0.05 mm to 0.1 mm, height from h = 0.1 mm to 0.4 mm, and gap from g = 0.3 mm to 0.4 mm increasing by every 0.1 mm. The
Fig. 7. a) Microscope and real-time observation software. b) Structure of arraydistributed soft magnets and distribution of magnetic beads. c) Structure of lattice-distributed soft magnets and distribution of magnetic beads.
concentration of magnetic bead mixture solution is c = 1%, and the density and average diameter of magnetic bead are ρ = 1.05 × 103 kg/ m3 and r = 2.5 μm respectively. The iron powders used in the experiment are made of ultrafine iron tetroxide with the average particle size of 10 μm and volume density of 1.9 g/cm3, and the relative permeability of the iron power is about 10. The fluid viscosity of magnetic beads mixture is 0.8937 pa·s. The distribution of magnetic beads can be observed in a microscope by removing the PMMA layer. Fig. 7b shows the distribution of magnetic beads captured with array-distributed soft magnets in the inlet
Fig. 6. a) Distribution of vertical component gradient mode of magnetic field intensity in lattice-distributed soft magnets. b) Distribution of magnetic beads in latticedistributed soft magnets. 6
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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velocity of vf = 0.03 m/s and height of h = 0.3 mm. The captured magnetic beads are present in the shape of strips and mostly concentrated on the magnetic stripes. According to the color depth and length of the stripes formed by the magnetic beads, the number of magnetic beads captured can be inferred. The magnetic beads are mainly captured at the edges of the magnetic strips near the inlet and gradually decrease along the direction from the inlet to the outlet. Besides, all of the magnetic beads are captured in the chamber, which is the same with the result of simulation in Fig. 5. Distribution of magnetic beads captured with lattice-distributed soft magnets is shown in Fig. 7c at the inlet velocity of vf = 0.03 m/s, radius of Rc = 0.1 mm, height of h = 0.3 mm and gap of g = 0.3 mm. Most of the magnetic beads are captured above the soft magnets and appear as a circle with the same radius of the soft magnets. The magnetic beadsand are completely captured in the chamber and gradually reduced from the inlet to the outlet, which is consistent with the result of simulation in Fig. 6. Compared with the array-distributed soft magnets, the latticedistributed soft magnets attract the magnetic beads farther away from the outlet of chamber, and the magnetic beads are more efficiently captured in the same conditions. Because the amount of the mixture passing through the chamber is kept in 2 ml per experiment, the ratio of the number of magnetic beads captured in different experiments can be approximated by the ratio of the number of pixels in different distribution images through binarization method. To study the effect of inlet flow rate on the capture efficiency of magnetic beads under the action of the soft magnets, several experiments are carried out with inlet velocities varying from 0.01 m/s to 0.05 m/s respectively, and the distribution of captured magnetic beads is displayed in Fig. 8. When the inlet velocity is 0.01 m/ s, the magnetic beads can be completely captured and the number of black pixels of distribution image is recorded as the replacement for the number of magnetic beads. As the inlet velocity increases to 0.03 m/s, the magnetic beads are captured at the soft magnets throughout the chamber and the soft magnets on the side of the chamber attract more magnetic beads. When the inlet velocity increased to 0.05 m/s, some of the magnetic beads escaped from the chamber, and the remaining magnetic beads are still absorbed by the soft magnets. For the simulations in this paper, the separation efficiency of the magnetic beads can be represented by ε, which is the ratio of the number of magnetic beads captured in the chamber to the total number of magnetic beads from the inlet. On the other hand, the separation efficiency in the experiments is replaced by the pixel ratio λ, which is the ratio of the numbers of binarized pixels in the magnetic bead distribution images at different speeds to the number of pixels at the velocity of 0.01 m/s. As shown in Fig. 9, the simulation results generally agree well with the experimental results, and the predicted and tested separation efficiencies have the same trend. When the inlet velocity is 0.01 m/s, the magnetic beads can be completely captured in the chamber. As the inlet velocity increases, the separation efficiency of the magnetic beads gradually decreases, but the rate of decreasing increases. However, when the inlet velocity is increased to 0.05 m/s, the
Fig. 9. Separation efficiency of magnetic beads with lattice-distributed soft magnets.
separation efficiency of the magnetic beads still maintains a relatively high level of about 80%. 6. Conclusions In summary, a microfluidic bioseparation chip that integrates soft magnets for magnetic separation applications is presented in the paper. The detection reagent in this microfluidic chip is mixed, heated and separated by two different volume chambers and integrated heating and magnetic devices, achieving the extraction of nucleic acid and protein molecules in the test sample. The array-distributed soft magnets and lattice-distributed soft magnets structures are designed respectively for the soft magnets, which are the key devices for magnetic beads separation, and the trajectories of the magnetic beads in these structures are simulated and verified by experiments. From the above experiment and investigations, some conclusions can be drawn. The separation effect of the magnetic beads is better when the soft magnets are distributed in lattice than the array-distributed soft magnets due to the increased gradient of the magnetic field intensity, and a significant increase in the magnetic field force can be achieved just by adjusting the local distribution of the soft magnets without changing the external magnetic field; the simulation and experimental results verify that the separation efficiency decreases with the velocity of flow rate and the lattice-distributed soft magnets maintain the separation efficiency of more than 80% when the inlet velocity increases from 0.01 m/s to 0.05 m/s, which provides the basis for the separation chamber as a connection of two consecutive
Fig. 8. Distribution of magnetic particles at different inlet velocity. 7
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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detection processes; the mature technologies are used in the fabrication of the bioseparation chip which are convenient to repeat the experiments. At the same time, there is still a certain limit to the increase of the magnetic field force by changing the distribution of soft magnets due to the limitation of the size of the magnets. It has an effect on the separation efficiency when the higher-speed fluid passes through. In this case, it is also necessary to increase the external magnetic field intensity to improve the separation efficiency in the future work.
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CRediT authorship contribution statement Yunfeng Zhu: Conceptualization, Methodology, Software, Resources, Data curation, Writing - original draft. Biao Zhang: Resources, Investigation. Jialiu Gu: Software, Data curation. Songjing Li: Supervision, Writing - review & editing, Validation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jmmm.2020.166485. References [1] I. Safarık, M. Safarıkova, Use of magnetic techniques for the isolation of cells, J. Chromatogr. B 722 (1999) 33–53, https://doi.org/10.1016/S0378-4347(98) 00338-7. [2] I. Safarik, M. Safarikova, Magnetic techniques for the isolation and purifcation of proteins and peptides, BioMagn. Res. Technol. 2 (2004) 7, https://doi.org/10. 1186/1477-044X-2-7. [3] M. Hejazian, N.T. Nguyen, Negative magnetophoresis in diluted ferrofluid flow, Lab Chip 15 (2015) 2998–3005, https://doi.org/10.1039/c5lc00427f. [4] T. Zhu, R. Cheng, S.A. Lee, E. Rajaraman, M.A. Eiteman, T.D. Querec, E.R. Unger, L. Mao, Continuous-flow ferrohydrodynamic sorting of particles and cells in microfluidic devices, Microfluid Nanofluid 13 (2012) 645–654, https://doi.org/10. 1007/s10404-012-1004-9. [5] A. Singh, S.K. Sahoo, Magnetic nanoparticles: a novel platform for cancer theranostics, Drug Discov. Today 19 (2014) 474–481, https://doi.org/10.1016/j.drudis. 2013.10.005. [6] X.L. Song, X.D. Luo, Q.Q. Zhang, A.P. Zhu, L.J. Ji, C.F. Yan, Preparation and characterization of biofunctionalized chitosan/Fe3O4 magnetic nanoparticles for application in liver magnetic resonance imaging, J. Mag. Mag. Mater. 388 (2015) 116–122, https://doi.org/10.1016/j.jmmm.2015.04.017. [7] M.L. Kovarik, D.M. Ornoff, A.T. Melvin, N.C. Dobes, Y. Wang, A.J. Dickinson, P.C. Gach, P.K. Shah, N.L. Albritton, Micro total analysis systems: fundamental advances and applications in the laboratory, clinic, and field, Anal. Chem. 85 (2013) 451–472, https://doi.org/10.1021/ac3031543. [8] Y.D. Peng, J.W. Nie, W.J. Zhang, J. Ma, C.X. Bao, Y. Cao, Magnetic particle chains embedded in elastic polymer matrix under pure transverse shear and energy conversion, J. Mag. Mag. Mater. 399 (2016) 116–122, https://doi.org/10.1016/j. jmmm.2019.02.078. [9] E.L. Jackson, H. Lu, Advances in microfluidic cell separation and manipulation, Curr. Opin. Chem. Eng. 2 (2013) 398–404, https://doi.org/10.1016/j.coche.2013. 10.001. [10] M.A.Md. Ali, K. Ostrikov, F.A. Khalid, B.Y. Majlis, A.A. Kayani, Active bioparticle manipulation in microfluidic systems, RSC Adv. 6 (2016) 113066–113094, https:// doi.org/10.1039/C6RA20080J. [11] M. Suwa, H. Watarai, Magnetoanalysis of micro/nanoparticles: a review, Anal.
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Research articles
Magnetic beads separation characteristics of a microfluidic bioseparation chip based on magnetophoresis with lattice-distributed soft magnets Yunfeng Zhu, Biao Zhang, Jialiu Gu, Songjing Li
T
⁎
Department of Fluid Control and Automation, Harbin Institute of Technology, Harbin 150001, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Microfluidics Magnetic bead Bioseparation chip Soft magnet Magnetic field Separation
To continuously perform pretreatment operations of biological detection reagents, a kind of microfluidic bioseparation chip that integrates mixer, heater and soft magnets for detection of nucleic acid and extraction of molecular proteins with magnetic beads methods is presented in the paper. A specific magnetic field is formed in a desired chamber bonded with PDMS and PMMA sheets by embedding soft magnets of iron powders in the chip, and magnetized by an external magnetic field. Soft magnets distributed in different ways are designed respectively, and the characteristics of the magnetic field, as well as the magnetic trajectory of the magnetic beads are simulated by considering the magnetic-fluid coupling. An experimental platform to observe the capturing of magnetic beads is built and the capture phenomenon for magnetic beads with lattice-distributed soft magnets is also experimentally verified. Based on the experimental data, the capture efficiency of the magnetic beads is calculated with the image processing method. The results show that lattice-distributed soft magnets are more effective than the array-distributed soft magnets for separating the magnetic beads. Furthermore, the latticedistributed soft magnets can improve the separation efficiency of the detected reagent maintained to be more than 80% when the inlet flow rate increases from 0.01 m/s to 0.05 m/s.
1. Introduction Sample pretreatment plays an important role in biomedical analysis for early diagnosis of diseases, detection of nucleic acid and extraction of molecular proteins. Routine phenol-chloroform method is a typical way of DNA extraction because it provides high-quality templates for amplification, but the step of proliferation in this method is makes this method time-consuming. The membrane adsorption method uses a silica gel membrane to absorb DNA with a high salt buffer solution and elutes DNA with a low salt TE buffer solution after washing several times. However, the disadvantage of easy pollution limited the application of membrane adsorption method. As a timesaving method of high purity, magnetic bead method became more and more widely used in nucleic acid extraction [1–4]. Considering that only few particles in the sample are paramagnetic, the immune magnetic beads made by the core-shell synthesis method with superparamagnetic material and polymer composite material covered were used as carriers for transport in reagent [5,6]. Covalently attached to magnetic beads by the functional groups such as amino group, carboxyl group, and thiol group, analyte particles were manipulated to separate, detect and purify genes, proteins, cells and microorganisms by applying an external magnetic field [7–10]. Usually, the processes of extracting test samples involve ⁎
reagent mixing, cracking, heating and eluting with reagents placed in centrifuge tubes and transferred to different equipments. To reduce the cost of using large inspection equipments, functional devices were more recently fabricated in microfluidic chips, which were an effective technique for miniaturization and integration [11]. In the process of sample pretreatment, magnetic bead separation has always been the focus of research as the crux in the extraction steps since the efficiency of separation directly affects the amount of extracted substance. For particles or cells in a microfluidic device to move magnetically, the magnetic field and its gradient are the main influencing factors. Permanent magnet (NdFeB) was placed near the microchannel to generate the magnetic field and field gradient for simple installation and mass manufacturing. Improvement of processing and manufacturing technology makes it possible to integrate high gradient microscale permanent magnets in microfluidic chips [12,13]. Preliminary experiment integrated two micro-permanent magnets on two sides of the microchannel and separately recorded the separation of the magnetic beads when the permanent magnets were installed with the same pole and opposite poles [14]. Optimization studies were conducted on the separation characteristics of the magnetic beads with circular permanent magnets distributed outside the circular flow channel [15,16]. Similar experiments conducted on separation of
Corresponding author. E-mail address: [email protected] (S. Li).
https://doi.org/10.1016/j.jmmm.2020.166485 Received 19 October 2019; Received in revised form 18 January 2020; Accepted 18 January 2020 Available online 21 January 2020 0304-8853/ © 2020 Elsevier B.V. All rights reserved.
Journal of Magnetism and Magnetic Materials 501 (2020) 166485
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Fig. 1. Structure of microfluidic bioseparation chip.
a microfluidic bioseparation chip with miniaturized and integrated devices with lattice-distributed soft magnets is proposed that implements the mixture of samples, heating of reagents, and separation of magnetic beads, to accomplish the whole process of detection continuously instead of multiple manual operations. Since the number of separated magnetic beads determines the number of protein molecules and nucleic acids available for detection, it is meaningful to explore an appropriate structure of soft magnets to enhance the separation efficiency of magnetic beads in this chip.
magnetic beads under the action of a non-micro permanent magnet integrated microfluidic chip [17]. In these experiments, magnetic particles or cells were driven by stored magnetic energy of permanent magnets without any external power, but attenuation and uncontrollability of magnetic field inevitably affect the flexibility of the microfluidic devices. In the other hand, magnetic forces of magnetic beads with an integrated micro permanent magnets were not sufficient for high flow rates, and the non-cancellable magnetic field affects the recovery of the separated magnetic beads. Electromagnets were also used to generate magnetic fields for the flexibility in changing the strength of the magnetic field by controlling the current. The initial study produced electromagnetic fields for magnetic bead control by integrating wires at a microchannel [18,19]. Subsequent researches were mainly aimed at the joule heating effect when an electric current passes through the integrated electromagnet by integrated cooling channel on silicon-based magnetron microfluidic chip or placing the micro-coil outside the chip [20]. However, whether changing the material and shape of the wire or integrating the cooling device on the chip significantly increases the difficulty and cost of the microfluidic chip fabrication, and cannot eliminate the influence of the thermal effect on the detection solution. In recent research, soft ferromagnetic was implanted in a microstructure to produce a high gradient and non-uniform magnetic field with external magnetic field. The scientific community paid more attention to the microfluidic bioseparation technology with an integrated soft magnet structure due to its advantages, such as larger magnetic field force range, excellent controllability and wider applicability. Several experiments have integrated soft magnets on the sides of the microchannels to produce greater magnetic field strength, but the distributions of magnetic fields are relatively complex [21,22]. In recent research, micro soft magnets were distributed on the sides of a T-shaped flow channel and a L-shaped flow channel [23]. The materials of soft magnets were investigated to increase the magnetic field force in the channel. However, there are few studies in the influence of soft magnet arrangement around microchannels on magnetic field forces. Since the microfluidic bioseparation chip was proposed, it has been the focus of researches to integrate soft magnet or micro magnet on the chips. At present, fabrication of micro magnet is still following the microfluidic chip technology to pattern the shapes combining photolithography with etching techniques in silicon chip or glass and obtain the metal layers by sputtering, electroplating, evaporation and vapor deposition [24–28]. According to the material used in the micro magnet, iron powder or NdFeB, soft magnet or permanent magnet can be formed after the external magnetic field is magnetized [29,30]. In the existing researches, scientists mainly focused on the movement of magnetic beads to simulate cells manipulation in blood vessels. However, during the whole process of biological test, magnetic beads not only pass through the rectangular microchannel but also are mixed and separated in several chambers with different volumes. In this paper,
2. Structure and working principle of the microfluidic bioseparation chip 2.1. Structure and fabrication of the chip In the pretreatment process of nucleic acid detection, the lysate is firstly mixed with the cells. After a nucleic acid is successfully lysed, it is adsorbed on a magnetic bead with the best effect at a specific temperature. After that, the magnetic beads with nucleic acids are separated in a specific chamber and the remaining solution is drained. The nucleic acids can be collected when the elution solution is mixed with the magnetic beads in the final. Therefore, the bioseparation chip is designed including the mixer, heater and separation chamber. The above process would be repeated several times for some different nucleic acids, so it is necessary to add another flow channels and chambers for mixing and heating after the separation chamber. In this condition, the efficiency of magnetic beads separation determines the number of magnetic beads in the subsequent processing and greatly influences the entire nucleic acids extraction and subsequent detection results. As shown in Fig. 1, the microfluidic bioseparation chip consists of two layers respectively made by Polydimethylsiloxane (PDMS) and Poly Methyl Methacrylatemethacrylic Acid (PMMA). The PDMS layer is mainly used for mixture, reaction, and separation of the magnetic bead solution. It is formed with two fluidic inlets and one waste outlet on the other side of the layer, connected with three microfluidic channels and two chambers. Two channels extending from the inlets intersect and form a curved channel which is connected to the heating chamber with a larger height than the channels. In contrast, the separation chamber is manufactured between the heating chamber and the waste outlet with the same height of the channels. This microfluidic device is fabricated at the bottom of the PDMS layer by traditional soft etching technique. In particular, the mold of microfluidic channels and the separation chamber is manufactured by dry films and the heating chamber is shaped by a cylindrical mold. The PMMA layer includes the heating element, the soft magnets and a thin PDMS film covering the upper surface. Computer Numerical Control (CNC) is used to mill the grooves for the heating device and soft magnets, and than a piece of special metal is embedded into the grooves as the heating device. The heating foil is electroplated directly under the heating chamber and soft 2
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Y. Zhu, et al.
linearly with the average magnetic field force of the magnetic beads at different positions in the chamber is used to describe the magnetic field force as
magnets are aligned with the separation chamber. Chosen as the material of soft magnets, iron powders are injected into the grooves milled by miniature engraving machine, and produce a gradient magnetic field in the separation chamber when they are magnetized by an external uniform magnetic field. A thin PDMS film is bonded to the PMMA layer by the surface treatment method, and it is bonded to the PDMS layer on other face.
1 Dh 0
P¯ =
h0
D
∫− D22 ∫− h2
0
→ → (H ·∇) H dxdz
2
The separation of the magnetic beads is mainly affected by the the magnetic field force component of in the vertical direction Fmz, which can be calculated by
2.2. Working principle Magnetic beads of micrometer or nanometer size are mixed with a dispersant to avoid agglomeration. The magnetic beads solution is injected into the microfluidic bioseparation chip and the sample reagent is poured into another inlet, keeping their quantities of flow in a constant ratio. Protein molecules or nucleic acids in the sample reagent are adequately mixed when flowing through the curved channel and adsorbed to functional groups on the surface of magnetic beads. The mixture from the curved channel is stored in the heating chamber which is designed to ensure the dose of test reagent and the biochemical reaction temperature through the heating foil under the chamber. After that, pulled by solution, the magnetic beads with the substance to be detected flow into the separation chamber to satisfy the requirement of subsequent eluent dosage. In this region, magnetic beads are affected by the magnetic field force of the soft magnets and change their directions of motion which were the same with the solution. Accordingly, the difference between two velocities causes fluid drag on magnetic beads to suppress the escape tendency of magnetic beads. When the magnetic beads are in micron scale, magnetic force, drag, gravity, and buoyancy are considered as the main forces in a magnetic-fluid coupling field, but the interaction between magnetic beads and Brownian force are ignored. The density of the magnetic beads used in this paper is similar to the solution, so the gravity and buoyancy of the magnetic beads can be neglected.
4μ0 πr 3χp
Fmz =
3 + χp
(Hx
4μ0 πr 3χp → ∂Hz ∂Hz ∂Hz + Hy + Hz )= (H ·∇Hz ) ∂x ∂y ∂z 3 + χp
(4)
It can be seen that the vertical component of the magnetic field force mainly depends on the magnetic field intensity H and the vertical component gradient of the magnetic field intensity ∇Hz . The permanent magnet that generates the external magnetic field is a cylindrical magnet with a radius of rm and a height of hm. If a coordinate system is created at the center of the bottom of the cylinder and (θ, zm) represents the point on the cylindrical surface, the components of the magnetic field intensity can be expressed as
Hx =
B0 dz m 4πμ0 μr
∫0 ∫0
2π
Hy =
B0 dz m 4πμ0 μr
∫0 ∫0
hm
2π
B0 dz m ( 4πμ0 μr
∫0 ∫0
hm
2π
hm
rm (z − z m) cos θ dθ K
(5)
rm (z − z m) sin θ dθ K
(6)
Hz =
−
hm
∫0 ∫0
2π
−rm (x − rm cos θ) cos θ dθ K
rm (y − rm sin θ) sin θ dθ ) K
K = [(x − rm cos θ)2 + (y − rm sin θ)2 + (z − z m)2]3/2 3. Calculation of magnetic force
(7) (8)
where θ is the angle between the x-axis and the perpendicular from the point to the z-axis; zm is the z coordinate of the point. The drag force coursed by velocity deviation of a magnetic bead Fd can be written as [33]
To study the trajectories of magnetic beads and the separation efficiencies of magnetic beads in the bioseparation chamber, the trajectory and magnetic field force of the magnetic beads in the above two soft magnets structures are respectively displayed by building the simulation models in a specific size. According to the simulation results, the separation efficiencies of magnetic beads in different magnetic fields are analyzed and compared.
→ Fd = 6πηr (→ vf − → vp) fD
(9)
where η and vf are the dynamic viscosity and velocity of the fluid respectively; fD is the hydrodynamic drag coefficient which is given by [34]
3.1. Effect of soft magnet
fD = [1 −
The magnetic force on a magnetic bead is given by [31]
4μ0 πr 3χp → → → Fm = (H ·∇) H 3 + χp
(3)
9 r 1 r 45 r 1 r ( )+ ( )3 − ( )4 − ( )5]−1 16 r + z ′ 8 r + z′ 256 r + z ′ 16 r + z ′ (10)
where z ′ is the distance between the magnetic bead and the surface of the microchannel. It can be seen that when the flow rate of the solution is higher or the magnetic bead are closer to the wall, the drag force of the magnetic bead Fd is larger. Then it will be more difficult to change the trajectory of this magnetic bead and capture it. As a result, the magnetic bead changes its trajectory due to these forces, either flowing to the drain channel with the solution or being adsorbed at the bottom of the chamber. The trajectory of the magnetic bead in the last movement fluctuation period before landing to the bottom is also displayed in Fig. 2. The direction of the magnetic field force points to the edge of the soft magnet at the beginning, with the magnetic bead moving down in the vertical direction. Then, the magnetic bead passes through the line 1 and the magnetic field force drags the magnetic bead upward until line 2 near the next soft magnet. After that, the magnetic bead is attracted to the second soft magnet again and finally captured at the edge where the magnetic field force is largest. In order to investigate the magnetic field force of the magnetic beads in the bioseparation chamber, several different magnetic field
(1)
where μ0 = 4π × 10−7 H/m is the magnetic permeability in the free space; r = 2.5 μm is the average diameter of the magnetic bead; χp = 0.2 is the magnetic volume susceptibility [32]; H is the magnetic field intensity at the center of magnetic bead. The following equation can be obtained from Eq. (1) as
→ → (H ·∇) H ∂Hy ∂Hy ∂Hy → → ∂H ∂H ∂H + Hz = i (Hx x + Hy x + Hz x ) + j (Hx + Hy ) ∂z ∂y ∂x ∂y ∂z ∂x → ∂Hz ∂Hz ∂Hz + k (Hx + Hy + Hz ) ∂x ∂y ∂z (2) It can be derived from Eqs. (1) and (2) that the magnetic force of the → → magnetic beads is linear with (H ∙∇) H when the material and size of the magnetic beads have been determined. A defined quantity that varies 3
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field intensity are presented in Fig. 3b to d respectively. It can be seen from Fig. 3b that under the effect of a single permanent magnet with a magnetization of 500,000 A/m, the magnetic field intensity in the chamber is from |H| = 4.9 × 104 A/m to |H| = 5.1 × 104 A/m. When two identical permanent magnets are placed on both sides of the chamber in the same polarity direction, the magnetic field intensity in the chamber is almost constant and remains at |H| = 1.01 × 105 A/m. When the soft magnets are placed between the two permanent magnets, the magnetic field intensity above the soft magnets decreases dramatically with the distance to the soft magnets in the vertical direction but it increases with the distance to the chamber in the region between the soft magnets. The maximum magnetic field intensity |H| = 1.31 × 105 A/m is displaced at two edges of the soft magnet, and the minimum magnetic field intensity |H| = 7.89 × 104 A/m appears in the center of the gap. It is shown in Fig. 3c that the vertical component gradient model of the magnetic field intensity changes between |∇Hz| = 7.94 × 106 A/m2 and |∇Hz| = 8.26 × 106 A/m2 with a single permanent magnet. However, the vertical component gradient model of the magnetic field intensity increases significantly from |∇Hz| = 1.54 × 104 A/m2 in the center of the chamber to |∇Hz| = 6.02 × 105 A/m2 in both ends along the horizontal direction when two permanent magnets are placed. After integrating the soft magnets, the vertical component gradient mode of the average magnetic field intensity increases from |∇Hz| = 3.32 × 107 A/m2 to |∇Hz| = 8.26 × 106 A/m2, and the maximum value locates at the outer ends of the soft magnet. The magnetic field force is displayed in Fig. 3d where the vertical compo→ nent of magnetic field force (H ·∇) Hz increases from −3.99 × 1011 A2/ 3 11 2 3 m to −4.16 × 10 A /m with a single permanent magnet and it changes symmetrically in the range of −1.17 × 1011 A2/m3 to 1.17 × 1011 A2/m3 with two permanent magnets. After integrating the → soft magnets, variable of vertical magnetic field force (H ·∇) Hz changes 13 2 3 13 2 3 from −8.22 × 10 A /m to 2.23 × 10 A /m , with the distribution characteristics similar to the magnetic field intensity, and the direction of magnetic field force in the range of the soft magnet is opposite to that between the soft magnets. According to the above magnetic field distribution, the magnetic field intensity generated by a single permanent magnet is the smallest, but the vertical component gradient model of the magnetic field intensity is much larger than that of the two permanent magnets, causing that magnetic field force is much larger than the latter. For the same reason, as the soft magnets are integrated between the two permanent magnets, the magnitude of the magnetic field intensity is not changed much, but the vertical component gradient model of the magnetic field intensity can be increased by hundreds of times, thereby greatly increasing the magnetic field force in the chamber. Therefore, the soft magnets mainly increase the magnitude of the magnetic field force by increasing the gradient of the magnetic field intensity.
Fig. 2. Forces and motion trajectory of magnetic bead between two adjacent soft magnets.
Fig. 3. a) Side view schematic diagram of single permanent magnet, double permanent magnets and permanent magnets with soft magnets beside the separation chamber. b) Magnetic field intensity in the chamber. c) The vertical component gradient mode of the magnetic field intensity. d) Distribution of variable of magnetic field force in the vertical direction.
3.2. Magnetic force in bioseparation chamber To analyze the force and motion of a magnetic bead in the combination of magnetic field and flow field intuitively, a 2D simulation model is built to simulate the magnetic field force and fluid drag force on single magnetic bead in COMSOL and the trajectory of the magnetic bead is drawn. Ten magnetic stripes with widths of w = 0.2 mm, heights of h = 0.5 mm, and gaps of g = 0.3 mm are magnetized by the external uniform magnetic field with magnetic flux density of B = 0.1 T. The separation chamber has the diameter of D = 5 mm and height of h0 = 0.2 mm. The distance between the soft magnets and the bottom of the chamber is hf = 0.04 mm. As shown in Fig. 2, the background magnetic field is superimposed with the magnetic field of soft magnets excited by itself, and magnetic flux density is drawn as isopotential curves. The magnetic flux density within the length range of the soft magnet is larger than that between two soft magnets because
generation methods are compared. The magnetic field distributions in the separation chamber with a single permanent magnet, double permanent magnets and integrated soft magnets shown in Fig. 3a are simulated by the software COMSOL. Ten soft magnets of the same shape are generated, and each magnet has the width of w = 0.2 mm, the height of h = 0.5 mm and the gap of g = 0.3 mm. The height of PDMS film is hf = 0.04 mm. For the permanent magnet, the width is 10 mm, the height is 2 mm and the distance to the channel is 1.5 mm. The separation chamber has a diameter of D = 5 mm and a height of h0 = 0.2 mm. The contours for the magnetic field force and magnetic 4
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Fig. 4. Schematic diagram of array-distributed soft magnets and lattice-distributed soft magnets.
mode of magnetic field intensity. It can be seen from the diagram that the gradient model of magnetic field intensity is the largest near the edge of a soft magnet and gradually decreases to both sides. There is a minimum of magnetic field intensity in the middle of a soft magnet and a smaller extremum between two adjacent magnetic stripes. In the whole separation chamber, the vertical component gradient mode of magnetic field intensity increases and decreases alternately along the flow direction. In particular, magnetic field intensity gradients of magnetic stripes on two sides of the soft magnets are remarkably larger than the others. As shown in Fig. 5b, the trajectory of the magnetic bead has a waving trend in the chamber, and the lowest points of each movement period appear at the back edges of the soft magnets. The magnetic beads generally move from the inlet to the outlet in the horizontal direction and present an up-and-down periodic motion in the vertical direction, eventually being captured near the edges of the soft magnets or escaping from the separation chamber. Along the direction from inlet to outlet in the chamber, the number of captured magnetic beads gradually reduces. When the magnetic beads entered the chamber, they are firstly subjected to the first magnetic stripe with the largest magnetic field intensity gradient. Therefore, the magnetic beads are mostly captured on the magnetic stripes in the front rows with a low inlet velocity.
the magnetic-field vector is in the same direction with the background magnetic field above the soft magnets and opposite in the gap. Obviously, the magnitudes and directions of the magnetic field forces only depend on the intensity of the magnetic field since the other physical parameters are constant. The direction of the magnetic field force is tangent to the direction of the magnetic field, that is, tangent to the magnetic induction line, and points from the low flux density to the high flux density. The magnetic field force vector distribution in the chamber is shown by the arrow in Fig. 2, where the magnetic field force is the largest at the edge and corner of the soft magnet, and gradually decreases at the surrounding space. 4. Design and calculation 4.1. Design of the lattice-distributed soft magnets The purpose of the design is to capture all the magnetic beads in the mixture at the bottom of the bioseparation chamber. In order to effectively enhance the magnetic field force and the intensity of magnetic field gradient, two distribution structures of soft magnets are displayed in Fig. 4. Array-distributed soft magnets consist of several stripe shape soft magnets fabricated from the inlet to the outlet of the bioseparation chamber, and the length of each soft magnet is the same with the diameter of the chamber. In the lattice-distributed structure, soft magnets are manufactured with multiple small cylinders and they are all confined within the bioseparation chamber. In particular, the number of columns of lattice-distributed soft magnets is always one more than the number of rows. The distance from the soft magnets to the bottom of the chamber is determined by the thickness of the PDMS film.
4.2.2. Distribution with lattice-distributed soft magnets According to the three dimensional simulation results in the last section, the gradient of magnetic field intensity is larger near the edges of the soft magnets. Thus, lattice-distributed soft magnets composed of small cylinders are implemented to increase the number of the soft magnet edges. Another three dimensional simulation model for soft magnets arranged at a matrix of 9 rows and 10 columns is built with the diameter of l = w = 0.2 mm, and gap of g = 0.3 mm. By keeping the remaining parameters unchanged, simulation results are drawn into diagrams of the trajectory of magnetic bead, magnetic field intensity gradient and distribution of captured magnetic beads in Fig. 6a and b. The average magnetic flux density mode in separation chamber is 0.11 T, which is similar to that of the array-distributed soft magnets, but vertical component gradient of magnetic field intensity is 1.527 × 108 A/m2, which is 18% larger than the former. Besides, the vertical component of the average magnetic field force is 1.115 × 10−12 N, 60% larger than that of the array-distributed soft magnets, whose average magnetic field force is 6.943 × 10−13 N. It can be seen from Fig. 6a that the gradient of the magnetic field intensity is larger near the edges of the soft magnets and the largest magnetic field intensity appears at the edge of every cylinder soft magnet, which is similar to array-distributed soft magnets. As shown in Fig. 6b, three magnetic beads are marked as examples for several typical trajectories in the separation chamber. The first magnetic bead passes through soft magnet directly after entering the chamber, and is captured rapidly at the edge of next soft magnet; the second magnetic bead has avoided the soft magnet at the beginning, and is still captured
4.2. Magnetic beads distribution 4.2.1. Distribution in array-distributed soft magnets A three dimensional model is also established to simulate distribution of the magnetic field in bioseparation chamber and trajectories of magnetic beads in this region. Parameters of simulation is the same with the 2D model and the length of magnetic stripe is l = 5 mm. Particle Tracing Module is chosen as the research method with three components which are Magnetic Fields No Currents (mfnc), Particle Tracing for Fluid Flow (fpt) and Laminar Flow (spf) respectively. The density and radius of particle are setting as 1000 kg/m3 and 2.5 μm, the relative permeability is 1000 and there are 300 particles ejected at the inlet. According to the results of numerical simulation, the average magnetic flux density mode in the bioseparation chamber increased by 0.01 T after the array-distributed soft magnets magnetized. However, the vertical component gradient model of the average magnetic field intensity increases from 0 to 1.297 × 108 A/m2, indicating that the soft magnets greatly enhance the gradient mode of the magnetic field intensity. Fig. 5a displays the distribution of vertical component gradient 5
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Fig. 5. a) Distribution of vertical component gradient mode of magnetic field intensity in array-distributed soft magnets. b) Distribution of magnetic beads in arraydistributed soft magnets.
when it moves to the region of the soft magnet by the fluid drag force; the third magnetic bead is pulled straightly along the gap between the soft magnets where the vertical component of the magnetic force of magnetic bead is upward and the magnetic bead is finally captured onto the upper surface of the chamber. From the trajectories of the above three particles, it can be seen that the position of the magnetic field where the magnetic bead passes through in the chamber affects the trajectory of the magnetic bead. Moreover, when the magnetic bead closer to the channel wall, the velocity of magnetic bead is lower, and the drag force of the magnetic bead would be more important, reducing the velocity of magnetic bead at the entrance of the chamber and trapping the magnetic bead quicker. The final distribution of magnetic beads captured in the chamber is also displayed in Fig. 6b, showing that magnetic beads are captured more evenly with lattice-distributed soft magnets compared to the array-distributed soft magnets. The number of magnetic beads that are captured in the middle of the chamber increases but less magnetic beads are pulled to the outlet. Moreover, it is different from the array-distributed soft magnets that the separation of the magnetic beads can be effectively separated in the vertical direction in both the region of the soft magnets and the gap between the soft magnets. 5. Experiment and discussion In order to facilitate the observation of the magnetic beads distribution in the chamber after separation, the surfaces of the microfluidic chip are sealed with two films respectively to remain these two layers detachable. To record the captured magnetic beads, microfluidic bioseparation chip with array-distributed soft magnets and lattice-distributed soft magnets are fabricated in PMMA plates with the thickness of 1 mm, and covered by the thin PDMS films with the thickness of 0.02 mm. The PDMS layer with channels and chambers is also covered by another thin PDMS films with the thickness of 0.02 mm. The structure of the soft magnets can be observed through a microscope connected to the computer which is shown in Fig. 7. The array-distributed soft magnets are manufactured with the length of l = 5 mm, the width of w = 0.2 mm, height from h = 0.1 mm to 0.4 mm increasing by every 0.1 mm, and gap of g = 0.3 mm. The lattice-distributed soft magnets are manufactured with the radius from Rc = 0.05 mm to 0.1 mm, height from h = 0.1 mm to 0.4 mm, and gap from g = 0.3 mm to 0.4 mm increasing by every 0.1 mm. The
Fig. 7. a) Microscope and real-time observation software. b) Structure of arraydistributed soft magnets and distribution of magnetic beads. c) Structure of lattice-distributed soft magnets and distribution of magnetic beads.
concentration of magnetic bead mixture solution is c = 1%, and the density and average diameter of magnetic bead are ρ = 1.05 × 103 kg/ m3 and r = 2.5 μm respectively. The iron powders used in the experiment are made of ultrafine iron tetroxide with the average particle size of 10 μm and volume density of 1.9 g/cm3, and the relative permeability of the iron power is about 10. The fluid viscosity of magnetic beads mixture is 0.8937 pa·s. The distribution of magnetic beads can be observed in a microscope by removing the PMMA layer. Fig. 7b shows the distribution of magnetic beads captured with array-distributed soft magnets in the inlet
Fig. 6. a) Distribution of vertical component gradient mode of magnetic field intensity in lattice-distributed soft magnets. b) Distribution of magnetic beads in latticedistributed soft magnets. 6
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velocity of vf = 0.03 m/s and height of h = 0.3 mm. The captured magnetic beads are present in the shape of strips and mostly concentrated on the magnetic stripes. According to the color depth and length of the stripes formed by the magnetic beads, the number of magnetic beads captured can be inferred. The magnetic beads are mainly captured at the edges of the magnetic strips near the inlet and gradually decrease along the direction from the inlet to the outlet. Besides, all of the magnetic beads are captured in the chamber, which is the same with the result of simulation in Fig. 5. Distribution of magnetic beads captured with lattice-distributed soft magnets is shown in Fig. 7c at the inlet velocity of vf = 0.03 m/s, radius of Rc = 0.1 mm, height of h = 0.3 mm and gap of g = 0.3 mm. Most of the magnetic beads are captured above the soft magnets and appear as a circle with the same radius of the soft magnets. The magnetic beadsand are completely captured in the chamber and gradually reduced from the inlet to the outlet, which is consistent with the result of simulation in Fig. 6. Compared with the array-distributed soft magnets, the latticedistributed soft magnets attract the magnetic beads farther away from the outlet of chamber, and the magnetic beads are more efficiently captured in the same conditions. Because the amount of the mixture passing through the chamber is kept in 2 ml per experiment, the ratio of the number of magnetic beads captured in different experiments can be approximated by the ratio of the number of pixels in different distribution images through binarization method. To study the effect of inlet flow rate on the capture efficiency of magnetic beads under the action of the soft magnets, several experiments are carried out with inlet velocities varying from 0.01 m/s to 0.05 m/s respectively, and the distribution of captured magnetic beads is displayed in Fig. 8. When the inlet velocity is 0.01 m/ s, the magnetic beads can be completely captured and the number of black pixels of distribution image is recorded as the replacement for the number of magnetic beads. As the inlet velocity increases to 0.03 m/s, the magnetic beads are captured at the soft magnets throughout the chamber and the soft magnets on the side of the chamber attract more magnetic beads. When the inlet velocity increased to 0.05 m/s, some of the magnetic beads escaped from the chamber, and the remaining magnetic beads are still absorbed by the soft magnets. For the simulations in this paper, the separation efficiency of the magnetic beads can be represented by ε, which is the ratio of the number of magnetic beads captured in the chamber to the total number of magnetic beads from the inlet. On the other hand, the separation efficiency in the experiments is replaced by the pixel ratio λ, which is the ratio of the numbers of binarized pixels in the magnetic bead distribution images at different speeds to the number of pixels at the velocity of 0.01 m/s. As shown in Fig. 9, the simulation results generally agree well with the experimental results, and the predicted and tested separation efficiencies have the same trend. When the inlet velocity is 0.01 m/s, the magnetic beads can be completely captured in the chamber. As the inlet velocity increases, the separation efficiency of the magnetic beads gradually decreases, but the rate of decreasing increases. However, when the inlet velocity is increased to 0.05 m/s, the
Fig. 9. Separation efficiency of magnetic beads with lattice-distributed soft magnets.
separation efficiency of the magnetic beads still maintains a relatively high level of about 80%. 6. Conclusions In summary, a microfluidic bioseparation chip that integrates soft magnets for magnetic separation applications is presented in the paper. The detection reagent in this microfluidic chip is mixed, heated and separated by two different volume chambers and integrated heating and magnetic devices, achieving the extraction of nucleic acid and protein molecules in the test sample. The array-distributed soft magnets and lattice-distributed soft magnets structures are designed respectively for the soft magnets, which are the key devices for magnetic beads separation, and the trajectories of the magnetic beads in these structures are simulated and verified by experiments. From the above experiment and investigations, some conclusions can be drawn. The separation effect of the magnetic beads is better when the soft magnets are distributed in lattice than the array-distributed soft magnets due to the increased gradient of the magnetic field intensity, and a significant increase in the magnetic field force can be achieved just by adjusting the local distribution of the soft magnets without changing the external magnetic field; the simulation and experimental results verify that the separation efficiency decreases with the velocity of flow rate and the lattice-distributed soft magnets maintain the separation efficiency of more than 80% when the inlet velocity increases from 0.01 m/s to 0.05 m/s, which provides the basis for the separation chamber as a connection of two consecutive
Fig. 8. Distribution of magnetic particles at different inlet velocity. 7
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detection processes; the mature technologies are used in the fabrication of the bioseparation chip which are convenient to repeat the experiments. At the same time, there is still a certain limit to the increase of the magnetic field force by changing the distribution of soft magnets due to the limitation of the size of the magnets. It has an effect on the separation efficiency when the higher-speed fluid passes through. In this case, it is also necessary to increase the external magnetic field intensity to improve the separation efficiency in the future work.
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CRediT authorship contribution statement Yunfeng Zhu: Conceptualization, Methodology, Software, Resources, Data curation, Writing - original draft. Biao Zhang: Resources, Investigation. Jialiu Gu: Software, Data curation. Songjing Li: Supervision, Writing - review & editing, Validation. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jmmm.2020.166485. References [1] I. Safarık, M. Safarıkova, Use of magnetic techniques for the isolation of cells, J. Chromatogr. B 722 (1999) 33–53, https://doi.org/10.1016/S0378-4347(98) 00338-7. [2] I. Safarik, M. Safarikova, Magnetic techniques for the isolation and purifcation of proteins and peptides, BioMagn. Res. Technol. 2 (2004) 7, https://doi.org/10. 1186/1477-044X-2-7. [3] M. Hejazian, N.T. Nguyen, Negative magnetophoresis in diluted ferrofluid flow, Lab Chip 15 (2015) 2998–3005, https://doi.org/10.1039/c5lc00427f. [4] T. Zhu, R. Cheng, S.A. Lee, E. Rajaraman, M.A. Eiteman, T.D. Querec, E.R. Unger, L. Mao, Continuous-flow ferrohydrodynamic sorting of particles and cells in microfluidic devices, Microfluid Nanofluid 13 (2012) 645–654, https://doi.org/10. 1007/s10404-012-1004-9. [5] A. Singh, S.K. Sahoo, Magnetic nanoparticles: a novel platform for cancer theranostics, Drug Discov. Today 19 (2014) 474–481, https://doi.org/10.1016/j.drudis. 2013.10.005. [6] X.L. Song, X.D. Luo, Q.Q. Zhang, A.P. Zhu, L.J. Ji, C.F. Yan, Preparation and characterization of biofunctionalized chitosan/Fe3O4 magnetic nanoparticles for application in liver magnetic resonance imaging, J. Mag. Mag. Mater. 388 (2015) 116–122, https://doi.org/10.1016/j.jmmm.2015.04.017. [7] M.L. Kovarik, D.M. Ornoff, A.T. Melvin, N.C. Dobes, Y. Wang, A.J. Dickinson, P.C. Gach, P.K. Shah, N.L. Albritton, Micro total analysis systems: fundamental advances and applications in the laboratory, clinic, and field, Anal. Chem. 85 (2013) 451–472, https://doi.org/10.1021/ac3031543. [8] Y.D. Peng, J.W. Nie, W.J. Zhang, J. Ma, C.X. Bao, Y. Cao, Magnetic particle chains embedded in elastic polymer matrix under pure transverse shear and energy conversion, J. Mag. Mag. Mater. 399 (2016) 116–122, https://doi.org/10.1016/j. jmmm.2019.02.078. [9] E.L. Jackson, H. Lu, Advances in microfluidic cell separation and manipulation, Curr. Opin. Chem. Eng. 2 (2013) 398–404, https://doi.org/10.1016/j.coche.2013. 10.001. [10] M.A.Md. Ali, K. Ostrikov, F.A. Khalid, B.Y. Majlis, A.A. Kayani, Active bioparticle manipulation in microfluidic systems, RSC Adv. 6 (2016) 113066–113094, https:// doi.org/10.1039/C6RA20080J. [11] M. Suwa, H. Watarai, Magnetoanalysis of micro/nanoparticles: a review, Anal.
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