Magnetic Separation of Malaria-Infected Red Blood Cells in Various

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Magnetic separation of malaria-infected red blood cells in various developmental stages Jeonghun Nam, Hui Huang, Hyungjung Lim, Chae-Seung Lim, and Sehyun Shin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac4012057 • Publication Date (Web): 01 Jul 2013 Downloaded from http://pubs.acs.org on July 6, 2013

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Analytical Chemistry

Magnetic separation of malaria-infected red blood cells in various developmental stages Jeonghun Nam, ‡a Hui Huang, ‡b Hyunjung Lim,a Chaeseung Lim,c and Sehyun Shin*a a

School of Mechanical Engineering, Korea University, 136-713, Seoul, Korea

b

Department of clinical laboratory science, Southwest Hospital, third military medical university,

Chongqing, China c

Department of Laboratory medicine, College of Medicine, Korea University Guro Hospital,

Seoul, Korea

ABSTRACT

Malaria is a serious disease that threatens the public health, especially in developing countries. Various methods have been developed to separate malaria-infected RBCs (i-RBCs) from blood samples for clinical diagnosis and biological and epidemiological researches. In this study, we propose a simple and label-free method for separating not only late-stage but early-stage i-RBCs on the basis of their paramagnetic characteristics due to the malaria by-product, hemozoin, by using a magnetic field gradient. A polydimethylsiloxane (PDMS) microfluidic channel was fabricated and integrated with a ferromagnetic wire fixed on a glass slide. To evaluate the performance of the microfluidic device containing the ferromagnetic wire, lateral displacement of NaNO2-treated RBCs, which also have paramagnetic characteristics, was observed with

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various flow rates. The results showed excellent agreement with theoretically predicted values. The same device was applied to separate i-RBCs. Late-stage i-RBCs (trophozoites and schizonts), which contains optically visible black dots, were separated with a recovery rate of approximately 98.3%. In addition, using an optimal flow rate, early-stage (ring-stage) i-RBCs, which had been difficult to separate due to the low paramagnetic characteristics, were successfully separated with a recovery rate of 73%. The present technique, using permanent magnets and ferromagnetic wire in a microchannel, can effectively separate i-RBCs in various developmental stages so that it may provide a potential tool for studying invasion mechanism of the malarial parasite as well as anti-malarial drug assays.

Introduction Malaria infection is a serious public health problem in the developing countries. The World Health Organization estimates 300-500 million clinical cases and more than 1 million deaths every year due to malaria infection. Pregnant women and children less than 5 years old are at the greatest risk of serious morbidity.1, 2 Human malaria is caused by 4 types of Plasmodium parasites; Plasmodium falciparum, P. malariae, P. ovale, and P. vivax. Among these, P. falciparum infection is the most fatal. Upon infection, P. falciparum parasites go through pre-erythrocytic and intra-erythrocytic stages in the human host. In the pre-erythrocytic stages, sporozoites, which are transmitted by mosquitoes, invade liver cells and produce thousands of merozoites, which can then invade red blood cells (RBCs).3-5 In the intra-erythrocyte stages, the infected red blood cells (i-RBCs) undergo various stages (ring, trophozoite and schizont) in a 48-hour cycle. At the end of the intra-erythrocyte


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stages, roughly 16-18 merozoites are produced within a schizont, which subsequently bursts to release the merozoites, leading to infection other RBCs.3-5 As this infection process repeats, the rate of infection of blood (the number of i-RBCs in the total volume of a blood sample) increases exponentially. Therefore, development of techniques for early diagnosis of i-RBCs at a low infection rate is necessary. For malaria diagnosis, the Giemsa-staining method has been widely used as the gold standard because of its high accuracy. However, the procedure for this method is complex, and welltrained personnel are required for reliable evaluation. On the other hand, paper-based test kits based on antigen-antibody reactions have been commercially developed.6-10 These test kits are simple to use and easily detect the existence of i-RBCs in blood, eliminating the need for a skilled technician. However, it is difficult to achieve high detection accuracy at low infection rates (approximately < 100 parasites/µL). Therefore, development of a precision i-RBC separation technique is required for clinical applications and malaria research.11 Separation of i-RBCs would enrich rare target cells from the low infection-rate sample solution and markedly enhance the accuracy of malaria diagnosis in a clinical setting. In particular, it is important to separate the newly-infected ring-stage i-RBCs, because, rather than late-stage i-RBCs, ring-stage i-RBCs circulate in the peripheral blood stream of malaria-infected patients.12-14 Separation of i-RBCs in various developmental stages has been requested for biological and epidemiological research, such as for anti-malarial drug assays15 and for studying the invasion mechanism of the malarial parasite.16 Even for the massive research needs, stage-synchronized separation of i-RBCs has not been successfully developed due similar properties of i-RBCs.


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Many of physical properties of i-RBCs, such as density, electrical conductivity, deformability, and magnetic properties, change owing to the metabolic changes that occur in the parasites during the various developmental stages. In addition, as the malaria parasites mature, the density of i-RBCs decreases.17 Taking the characteristics into account, various methods have been developed for concentration and separation of i-RBCs. A density gradient separation method using Percoll has been commonly used.11 However, this method requires an expensive working fluid (Percoll) and involves a complex process for preparation of the density gradient. Using the fact that the electrical conductivity of the i-RBC membrane increases with the progressing developmental stages.13,

18

Gascoyne et al. developed a dielectrophoretic separation device,

which utilized using the electrical differences between i-RBCs and healthy RBCs for separation.13, 18 However, for effective separation with this device, a buffer medium with low electrical conductivity must be used. Conventional magnetic-activated cell separation (MACS) on a magnetic beads column can also be used for label-free i-RBC separation based on their paramagnetic differences with healthy RBCs.19-22 Malaria parasites live by feeding off the hemoglobin in RBCs. Through polymerization and oxidation, the parasites convert the hemoglobin, which is toxic to the parasites, into an insoluble crystal known as hemozoin. The iron (Fe3+) in hemozoin has a stronger paramagnetic effect than the iron in hemoglobin (Fe2+). Therefore, i-RBCs usually behave as paramagnetic particles in a magnetic field.21-23 However, a macro-scale magnetic separation technique, which has large-scale dimensions, cannot exert an effective magnetic force on RBCs. In particular, in the case of ring-stage i-RBCs, low paramagnetic characteristics due to the rarity of hemozoin limits the separation capability of conventional magnetic separation techniques.24


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Recently, Hou et al. developed a microfluidic device for deformability-based separation of iRBCs inspired by the in vivo phenomenon of cell margination.14, 25 As maturation of i-RBCs proceeds, the innate deformability of the RBCs decreases.14, 26, 27 Using this microfluidic device, high-throughput separation can be achieved without any external forces. However, the purity of the separated i-RBCs may be low because of high hematocrit, which is an essential prerequisite for margination. To overcome the limitations of conventional separation of i-RBCs, a continuous separation technique using microfluidics has been developed. Recently, using the ferromagnetic microstructure in the external magnetic field of permanent magnets, high magnetic field gradient was generated in the microchannel.28-30 However, despite the state-of-the-art technology of magnetophoretic separation and because of small native magnetic properties, the separation efficiency needs further improvement; therefore, most of the magnetic separation methods employ magnetic beads labeled with biological cells.28-32 Magnetic labeling has inherent disadvantages; requiring complex sample preparation and therefore, additional time and expense. It also carries a risk of causing cell damages. More recently, Han et al. developed a diagmagnetic/ paramagnetic magnetophoretic microseparator.33-35 This magnetophoretic system, containing a ferromagnetic nickel structure and a permanent magnet, was able to separate red and white blood cells from diluted whole blood without magnetic tagging by using the high magnetic field gradient generated in the microchannel. However, no research results are successful for continuous, label-free separation of i-RBCs in ring-stages as well as late stages. In this paper, we introduce an innovative design for a microfluidic system to separate i-RBCs from a cultured malaria sample by using the magnetic properties of accumulated hemozoin in i-


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RBCs. Prior to separation of i-RBCs, the exposure time of RBCs in a high magnetic field gradient was calculated theoretically. The primary objective of the study is to develop a continuous, label-free separation method for not only late-stage but early-stage i-RBCs, focusing on the potential use of a magnetic separation device as a research tool. Enrichment of i-RBCs in a simple microfluidic device would significantly enhance the accuracy of malaria diagnosis in clinical applications. Notably, stage-synchronization of cultured i-RBCs can be achieved by adjusting force balance between magnetic force and inertia, which can be controlled by varying flow rate in a fixed magnetic field gradient.

Experimental Section Working principle Fig. 1(a) shows a schematic of the separation of i-RBCs in the current study. The microchannel consists of 3 inlets and 2 outlets. First, the sample fluid, which is a mixture of healthy RBCs and i-RBCs, is injected from the center inlets and the sheath fluids are injected from the 2 side inlets. Adopting sheath flow from the side inlets allows all the RBCs of the sample fluid to be focused at an optimal distance from the nickel wire for initialization at the entrance, as shown in Fig. 1 (region ii). As RBCs flow in the microchannel, i-RBCs are forced to move towards the nickel wire by the above-described working principle. Due to the high gradient magnetic field in the main channel, paramagnetic i-RBCs tend to migrate toward the nickel wire, whereas healthy RBCs keep flowing along the focused streamline, as shown in Fig. 1 (region iii). At optimal flow rates of the sample and sheath fluids, i-RBCs can be separated and collected from the lower outlet, as shown in Fig. 1 (region iv).


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Figure 1. Malaria-infected RBC separation using high magnetic field gradient. (a) Schematic diagram of i-RBC separation using the paramagnetic characteristics of hemozoin in i-RBCs. (b) Working principle of the magnetophoretic separation with a ferromagnetic nickel wire in external magnetic field. (c) Photograph of the permanent magnet for applying external magnetic field in the microchannel and the microfluidic device consisting of the PDMS microchannel and a nickel wire.

A uniform external magnetic field, H0, applied normal to the ferromagnetic nickel wire, generates a high magnetic field gradient. Therefore, RBCs flowing near the nickel wire experience a magnetic force due to the high magnetic field gradient. In Cartesian coordinates, the magnetic force of a rectangular wire on RBCs near the nickel wire can be calculated as35, 36 ∆χV μ

(1)

where ∆χ (=χRBC-χBuffer) is the relative magnetic susceptibility of an RBC with respect to the buffer solution; VBC is the volume of the RBC; µ0 is the magnetic susceptibility of the air; Ms is


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the saturation magnetization field of the wire; a is the radius of the circular ferromagnetic wire; x and y are Cartesian coordinates; H0 is the external magnetic field; and !a" is the unit vector in the x-direction in Cartesian coordinates. For low Reynolds numbers, the lateral direction velocity of the RBCs in the high magnetic field gradient can be expressed as:36 #

$%&

(2)

) *

'(

where η is the apparent viscosity of the buffer solution; A is the maximum cross-section area perpendicular to the main flow direction; and l is the characteristic length of the blood cells in the flow direction. Using equations (1) and (2), if the RBC is assumed to move only in the direction of the microchannel width, the trapping time, or exposure time to the magnetic field gradient, required for an RBC to move from the (x1, y) position to the (x2, y) position, (where +' , + ), can be calculated as: t ∆/0

) *

. (

%& . 1

2+'3 +3 4 267 8 +' + 4 28 3 4 217 3 98 7 ; ?

@

?

? 4 ? ? A

where K

. 1 C

>

(3)

.

From the extent of the magnetic susceptibility and lateral displacement of RBC, the exposure time required for separating cells in the magnetic field gradient can be calculated theoretically. Fig. 1(b) shows the working principle of the magnetophoretic separation with a nickel wire. A uniform external magnetic flux applied from a permanent magnet was deformed near the nickel wire, which generates a high gradient magnetic field. i-RBCs were drawn closer to the nickel


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wire due to the paramagnetic characteristics of hemozoin in i-RBCs, whereas healthy RBCs flowed along the initial position without lateral migration.

Device fabrication As shown in Fig. 2, a polydimethylsiloxane (PDMS) microfluidic channel, 50 µm in height and 100 µm in width, was fabricated on a replica mold, which was fabricated on a silicon wafer through photolithography by using an SU-8 negative photoresist (MicroChem, Newton, MA). The base and curing agent of PDMS were mixed, degassed in a vacuum chamber, and cured in an oven (80°C for 1h). A 50 µm diameter nickel wire (Alfa Aesar) was fixed on a glass slide using tape.

Figure 2. Schematic diagram of the fabrication process. a Fabrication of the PDMS microchannel through the standard soft-lithography technique. b Punching holes for three inlets and two outlets. c Fixation of the nickel wire on the glass slide with adhesive tape. d Alignment of the PDMS microchannel and the nickel wire using high-purity ethanol after oxygen plasma treatment. e Drying of extra ethanol on the hot plate at 80 ℃ for 90 min and blocking of holes at both sides of PDMS microchannel with epoxy glue.


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The device for magnetic separation of i-RBC, consisting of the PDMS microchannel and the glass slide with the nickel wire, was bonded using the process shown in Fig. 2. Briefly, the PDMS microchannel and the glass slide with the nickel wire were treated with oxygen plasma at 250 W and 80 mTorr (CUTE, Femto Science Co., Korea) for 50 s. After oxygen plasma treatment, the PDMS slab and the glass slide with the nickel wire were dipped in high-purity ethanol (99.9%) to prevent instant, irreversible bonding and to allow smooth movement between each layer during alignment of the microchannel and the nickel wire. The residual ethanol remaining in the microchannel was dried on a hot plate at 80 ℃ for 90 min for irreversible bonding. Finally, holes on both sides of the PDMS microchannel were blocked with epoxy glue.

P.Falciparum culture Laboratory line 3D7 P. falciparum malaria parasites were grown with human erythrocytes (group O, Rh-positive, 3% hematocrit) in RPMI-HEPES medium supplemented with 40 mg/l Gentamicin (Invitrogen Co., USA), 1.36 g/l Hypoxanthine (Sigma Aldrich, USA), 25 mM HEPES (Sigma Aldrich, USA), 7.5 % Sodium Bicarbonate (Invitrogen Co., USA), 20 % Glucose (Sigma Aldrich, USA), 1 M NaOH (Sigma Aldrich, USA), and 20% Albumax (Invitrogen Co., USA). All cultures were maintained at 37℃ in an atmosphere of 5% CO2, 1% O2, and 94% N2, with daily medium changes.37 Synchronization of culture was achieved through sorbitol lysis at mature stage using 5 % sorbitol (Sigma Aldrich, USA), and fine-tuned by another lysis after 8 hours.38 Cells were harvested after cultured for 48 hours.


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NaNO2-treated RBC sample preparation For preparation of NaNO2-treated blood cells, blood samples were obtained from healthy volunteers, who were not on any medication and who provided informed consent. A venous blood sample was drawn from the antecubital vein and collected in (K2) EDTA (as anticoagulant) vacuum tubes (Vacutainers, 6 mL, BD, Franklin Lakes, NJ). The blood sample was mixed with 5 mM NaNO2 solution in a ratio of 1:40 (v/v) to oxidize the hemoglobin in the RBCs into paramagnetic form, and then kept at 4 °C for 40 min before use.39, 40 With the NaNO2 treatment of the RBCs, the ferrous ions (Fe2+) were converted to the ferric (Fe3+) state. The conversion can be regarded in the same way as the paramagnetic changes of i-RBCs.

Experimental setup A permanent magnet was used to create an external magnetic field of 0.6 T and a syringe pump was used to drive the fluid. The magnetic susceptibility of uninfected, healthy RBCs, NaNO2treated RBCs and late schizonts i-RBCs, relative to the buffer solution, can be determined as ∆χ EFGHIEJ K 0.01 ; 10N , ∆χOOPQRSQST K 3.9 ; 10N and ∆χVK 1.8 ; 10N , respectively.23, 41-44 The differences of magnetic susceptibility among various RBCs could be utilized to separate them with applying magnetic force. In addition, any small difference of magnetic susceptibility can be amplified with adopting magnetic field gradient using a nickel wire in a microchannel. Before sample injection, surface treatment with 1% bovine serum albumin (BSA) was performed to prevent cell adhesion to the channel walls.45 Blood cells including healthy RBCs and i-RBCs were suspended in RPMI medium with a concentration of 10% (v/v) and then injected at the center inlet and focused hydrodynamically by 2 layers of


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sheath flow. Blood cells were focused at 35 µm from the surface of the nickel wire. With controlling the focusing location of sample and total flow rate can separate i-RBCs in earlystages as well as late stages.

Giemsa-staining for analysis Uniformly dispersed, thin blood films were prepared from a blood sample after separation and stained with 10% (v/v) Giemsa solution. Giemsa-stained blood films were examined using an optical microscope. Separation efficiency was calculated as the percentage of each cell population from each outlet out of the total number of RBCs from the uniformly dispersed Giemsa-stained blood films. RBCs were categorized as healthy RBCs, ring stage i-RBCs, or latestage i-RBCs (trophozoites and schizonts). Under microscopic examination, each developmental stage could be identified.22 Ring-stage i-RBCs are ring-shaped with a light-purple cytoplasm, whereas late-stage i-RBCs have more than 1 nucleus and visible brown pigments.

Results and discussion To evaluate the separation performance of the microfluidic device, preliminary tests were performed using NaNO2-treated RBCs. Fig. 3(a) shows normalized displacements of NaNO2treated RBCs as a function of the flow rate. Through the whole experiment, even though the total flow rate was adjusted in a wide range, the ratio of the sample flow rate to sheath flows was kept constant (Qsheath1 : Qsample : Qsheath2=2:1:3). The focused stream width remained constant with fixed flow rate ratio owing to liquid single-phase flow focusing.46 The lateral displacements of the RBCs were measured from the images taken at the end of the main microchannel, which was


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30 mm downstream from the inlet where the cells were initially exposed to the high magnetic field gradient. In Fig. 3, the horizontal axis represents the applied flow rate, which is the inverse of the time for which blood cells were exposed to the high magnetic field gradient. In Fig. 3(b), the vertical axis represents the normalized displacement (y/Y), where y is the length of the cell displacement due to the magnetic field, and Y is the distance between the initially focused line and the surface of the nickel wire. Fig. 3(a) shows that the control group of healthy RBCs (hRBCs) without NaNO2 treatment was not influenced by the magnetic field, and thus did not show any detectable displacement from the initial position under a wide range of flow rates. The error bars in Fig. 3(a) indicate the thickness of the RBCs streamline which represent the sum of initial width of the focused stream and diffusion length under flow. Fig. 3(b) (case i) shows that the thickness was within ± 5 µm of the standard deviation. For NaNO2-treated RBCs with relatively high flow rate (> 3.6 µL min-1), the NaNO2-treated RBCs showed a slight lateral migration from the initial streamline, due to a short exposure time to the magnetic field gradient, as shown in Fig. 3(b) (case ii). The theoretical results for lateral displacements, depicted as a solid line in Fig. 3(a), were calculated with the relative magnetic susceptibility of fully-deoxygenated hemoglobin and exposure time (total flow rate). As shown in Fig. 3(a), normalized displacement decreased with flow rate, which is the inverse of the exposure time. In Fig. 3(b) (case iii), NaNO2-treated RBCs showed 38% of the normalized displacement (y/Y), which means that treated cells migrated 13 µm from the initial focused line at 1.6 µL min-1. In the low flow rate region below 1.2 µL min-1, NaNO2-treated

RBCs

reached

100%

of

normalized

displacement

theoretically

and

experimentally. Therefore, at low flow rates, NaNO2-treated RBCs adhered to the surface of the nickel wire as shown in Fig. 3(b) (case iv) because the exposure time was longer than 3.1 s.


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Figure 3. Lateral migration of healthy (h-) RBCs and NaNO2-treated RBCs in a microchannel. (a) Normalized displacement of h-RBCs and NaNO2-treated RBCs at 30 mm beyond where RBCs began to be exposed to the magnetic field gradient. Theoretical values, as a function of the total flow rate, were calculated using the relative magnetic susceptibility of fully deoxyhemoglobin. (b) RBCs, initially flowing along the hydrodynamically focused line (i), show different lateral migration patterns according to the flow rate (ii-iv).


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The experimental values of NaNO2-treated RBCs showed good agreement with the theoretical results. Experimental results were slightly less than the theoretical results. This discrepancy arises from the circular shape of the nickel wire, which causes a non-uniform magnetophoretic force in the vertical direction of the microchannel. In brief, the magnetic field gradient passing through the center of the circular nickel wire is higher than the magnetic field passing through the bottom of the wire. Therefore, cells in different initial locations experience different levels of magnetophoretic force. Therefore, cells placed in the middle of the microchannel were exposed to higher magnetic field gradient than those on the bottom of the microchannel. With the relative magnetic susceptibility of late-stage schizont i-RBCs,23 the optimal flow rate range for magnetophoretic separation was determined as 0.6-1.7 µL min-1 from the theoretical value. Based on the theoretical values, flow rates ranging from 0.1 µL min-1 and 2.0 µL min-1 were applied to separate i-RBCs in various developmental stages. At high flow rate (1.6 µL min1

), none of the RBCs, including h- and i-RBCs were attracted to the nickel wire, and all RBCs

continued flowing along the focused line, which could be due to the high flow inertia and the short time of exposure to the high magnetic field gradient (data not shown). As shown in Fig. 4(b), at a slightly decreased flow rate of 1.2 µL min-1, RBCs with opticallyvisible black dots showed lateral migration toward the nickel wire and flowed through outlet B, whereas RBCs without black dots continued to flow along the focused line. Notably, the hemozoin in i-RBCs from the trophozoite stage is optically visible with a microscope.24 The iRBCs, which were attracted to the nickel wire were expected to be in late developmental stages such as the trophozoites and schizonts stages. As described earlier, the magnetic force applied to cells in a high magnetic field gradient is proportional to the relative magnetic susceptibility of the cells, which is in turn related to the developmental stages. The i-RBCs, which have higher


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magnetic susceptibilities than h-RBCs, were drawn closer to the nickel wire in the main channel, whereas other cells were not affected significantly by the magnetic field gradient under the applied flow rate condition. Therefore, as shown in Fig. 4(c) and (d), RBCs collected at outlet A were expected to be h-RBCs or i-RBCs in early developmental stages, because of the absence of notable black dots in the RBCs, whereas RBCs with visible black dots (hemozoin) in outlet B were expected to be i-RBCs in late developmental stages, such as trophozoites and schizonts.


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Figure 4. (a) The recovery rate at each outlet for the collected sample after separation. Recovery rates were expressed as mean ± standard deviation (SD) from 5 repeated experiments. (b) At bifurcated outlet, healthy RBCs or early-stage i-RBCs continued flowing along the initial focused line, while late-stage i-RBCs with visible black dots were attracted toward the nickel wire. Separation of i-RBCs with optically-visible black dots is shown in the Supporting Information. (c-d) The distribution of collected RBCs at outlet A and B after separation respectively. At outlet A, healthy or early-stage infected RBCs were collected. Almost all the late-stage i-RBCs with black dots, which were expected to be hemozoin, were collected at outlet B.

To evaluate the separation efficiency at 1.2 µL min-1, RBCs collected from each outlet were analyzed based on optical microscopic images. The recovery rate is defiedn as the ratio of output sample to input sample. However, if there are no missing cells between inlet and outlets, then the recovery rate can be defined as a ratio of the number of target cells in a particular outlet to the total number of cells in the collected sample. In fact, there was no cell loss in the device due to sticking or adhesion of cells to the channel walls, since the microchannel was washed with bovine serum albumin (BSA). Thus, the total number of cells in the collected sample can be regarded as same as the number of input cells. Similar defition of the recovery rate have been used in previous researches.47-48 A graph of the recovery rates is shown in Fig. 4(a). Late-stage iRBCs were notably attracted toward the nickel wire due to the high magnetic field gradient and collected at outlet B with a recovery rate of 98.3%. Healthy RBCs and early stage i-RBCs, which continued flowing along the initial focused line, were collected at outlet A with a recovery rate of 92%.


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To provide a long exposure time in a fixed magnetic field gradient of RBCs, the flow rate was further decreased and the separation of i-RBCs was examined. At a flow rate of 0.8 µL min-1, hRBCs continued flowing along the initial focused line, while some i-RBCs with black dots were attracted and almost adhered to the nickel wire (data not shown). During the separation, some RBCs without optically-visible black dots, which could not be identified as either i-RBCs or hRBCs, were attracted to the nickel wire and collected at outlet B. To identify the unidentified RBCs collected from each outlet and verify the separation efficiency, cells were stained and counted. The standard Giemsa-staining method was used with uniform dispersion of collected RBCs on the glass slide, and approximately one thousand Giemsa-stained RBCs on the glass slides were counted. As shown in Fig. 5(b), Giemsa-stained RBC distribution at outlets A and B showed that iRBCs were successfully separated from mixed blood samples containing h-RBCs, and ring- and late-stage i-RBCs. Among the Giemsa-stained cells, ring-stage i-RBCs have a ring-shaped lightpurple cytoplasm, whereas trophozoites and schizonts have one or more purple nuclei and golden brown hemozoin pigments in the RBCs. Even RBCs without optically-visible black dots were separated and collected at outlet B. Separated ring-stage i-RBCs were found to be more developed after invasion than unseparated ring-stages.


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Figure 5. Separation of i-RBCs with optimal flow rate 0.8 µl min-1. (a) The recovery rate at each outlet for the collected sample after separation of i-RBCs without magnet as a control and with magnet. The error bars represent the standard deviation (SD) of average recovery rate from 5 repeated experiments. (b) Giemsa-stained RBCs at outlet A and B. i-RBCs which are labeled with black arrows are ring-stage i-RBCs, and other i-RBCs with one or more hemozoin pigments in the RBCs are late stage i-RBCs.


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Fig. 5(a) describes the recovery rate of RBCs at each outlet. At outlet B, the recovery rate was 99.2% for late-stage i-RBCs (trophozoites and schizonts) and 73% for ring-stage i-RBCs. The purity is defined as the ratio of the number of target RBCs to the total number of cells found at target outlet. Here, the target RBCs were i-RBCs in each stage and the target outlet was outlet B. At the optimal flow rate (0.14 µl/min), the purities of i-RBCs in ring-stage and the late-stage collected at outlet B were both nearly 99.9%. On the other hand, h-RBCs were not affected by the high magnetic field gradient and were recovered at outlet A with a rate of 100%. 27% of ring-stage i-RBCs collected at outlet A can be considered an error occurring due to reasons such as the initial focused location in the vertical direction and the slight difference in magnetic susceptibilities. To verify the magnetic effect on the separation of the ring-stage i-RBCs collected at outlet B, an identical separation experiment was conducted without the permanent magnets, which meant no external magnetic field was applied. Without a magnetic field gradient generated in the microchannel, all of the RBCs continued flowing along the initial focused line and were collected at outlet A, as shown in Fig. 5(a). Therefore, it can be said that even ringstage i-RBCs, which have small magnetic susceptibilities, can be affected by the high magnetic field gradient in the microchannel and separated. For the separation of i-RBCs with optimal flow rate, the throughput was about 1.4 ⅹ103 cells/s. Despite of the significance of separation of ring-stage i-RBCs,12-14 conventional magnetic separation methods could not separate them due to their low magnetic susceptibility. However, the present results successfully separate ring-stage i-RBCs from h-RBCs with adopting high gradient of magnetic field using a Nickel wire in a microfluidics. Further study with optimal design enables to separate the infected RBCs with respect to their stages with enhanced


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separation efficiency. Optimal study includes the initial focusing of i-RBCs, the design of bifurcated microchannels at the outlet, and other factors for separation of ring-stage i-RBCs.

Conclusion We have demonstrated a simple, label-free method for separating i-RBCs using a high magnetic field gradient in a continuous flow mode. The essential features in the current research are the high separation efficiency and the simple fabrication of the device with ferromagnetic nickel wire. Using the paramagnetic property due to hemozoin, late-stage i-RBCs (trophozoites and schizonts) can be laterally driven toward the nickel wire by the high magnetic field gradient and separated with an efficiency of approximately 99.2%. We also confirmed that the current technique enabled the separation of ring stages which could not be separated using conventional magnetic separation systems. Further improvement in separation efficiency may be achieved by considering the initial distance of i-RBCs from the nickel wire and optimizing the design of the bifurcated outlet microchannel. The features of the current separation method make it potentially-useful in blood sample preparation for clinical diagnosis of malaria. In addition, it could be useful in biological and epidemiological research.

ASSOCIATED CONTENT This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +82-2-3290-3377. Fax: +82-2-928-5825.


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Author Contributions ‡These authors contributed equally. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT The work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2009-0080636). This research was supported by Nano·Material Technology Development Program (Green Nano Technology Development Program) through the National Research Foundation of Korea(NRF) funded by the Ministry of Education, Science and Technology (No. 2011-0020090).

REFERENCES (1) (2) (3) (4) (5) (6) (7) (8) (9)

Organization, W. H., New perspectives malaria diagnosis, The Organization, 2000. Snow, R. W., Guerra, C. A., Noor, A. M., Myint, H. Y. and Hay, S. I., Nature, 2005, 434, 214-217. Cowman, A. F. and Crabb, B. S., Cell, 2006, 124, 755-766. Sauerwein, R. W., Roestenberg, M. and Moorthy, V. S., Nature Reviews Immunology, 2010, 11, 57-64. Wirth, D. F., Nature, 2002, 419, 495-496. Craig, M. H. and Sharp, B. L., Transactions of the Royal Society of Tropical Medicine and Hygiene, 1997, 91, 279-282. Jelinek, T., Grobusch, M., Schwenke, S., Steidl, S., Von Sonnenburg, F., Nothdurft, H., Klein, E. and Löscher, T., Journal of Clinical Microbiology, 1999, 37, 721-723. Makler, M., Palmer, C. and Ager, A., Annals of tropical medicine and parasitology, 1998, 92, 419-433. Martinez, A. W., Phillips, S. T., Carrilho, E., Thomas III, S. W., Sindi, H. and Whitesides, G. M., Analytical Chemistry, 2008, 80, 3699-3707.


22

Page 23 of 24

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32) (33) (34) (35) (36) (37) (38) (39)

Martinez, A. W., Phillips, S. T., Whitesides, G. M. and Carrilho, E., Analytical chemistry, 2009, 82, 3-10. Miao, J. and Cui, L., Nature protocols, 2011, 6, 140-146. Demirev, P., Feldman, A., Kongkasuriyachai, D., Scholl, P., Sullivan Jr, D. and Kumar, N., Analytical chemistry, 2002, 74, 3262-3266. Gascoyne, P., Mahidol, C., Ruchirawat, M., Satayavivad, J., Watcharasit, P. and Becker, F. F., Lab Chip, 2002, 2, 70-75. Hou, H. W., Bhagat, A. A. S., Chong, A. G. L., Mao, P., Tan, K. S. W., Han, J. and Lim, C. T., Lab Chip, 2010, 10, 2605-2613. Liu, S., Mu, J., Jiang, H. and Su, X., The American journal of tropical medicine and hygiene, 2008, 79, 178-184. Carmenza, S., Lucia, G. and Karen, K., Malaria Journal, 2011, 10. Wahlgren, M., Berzins, K., Perlmann, P. and Björkman, A., Clinical and experimental immunology, 1983, 54, 127. Gascoyne, P., Pethig, R., Satayavivad, J., Becker, F. F. and Ruchirawat, M., Biochimica et Biophysica Acta (BBA)-Biomembranes, 1997, 1323, 240-252. Bousema, J. T., Gouagna, L. C., Drakeley, C. J., Meutstege, A. M., Okech, B. A., Akim, I. N. J., Beier, J. C., Githure, J. I. and Sauerwein, R. W., Malaria Journal, 2004, 3, 18. Paul, F., Roath, S., Melville, D., Warhurst, D. and Osisanya, J., Lancet, 1981, 2, 70. Ribaut, C., Berry, A., Chevalley, S., Reybier, K., Morlais, I., Parzy, D., Nepveu, F., Benoit-Vical, F. and Valentin, A., Malaria Journal, 2008, 7, 45. Sebastian, B., Annette, O., Sangdao, S., Panudda, S., Peeranad, P., Pattamawan, C., Prida, M., Kovit, P. and Hartmut, N., Malaria Journal, 2010, 9, 38. Moore, L. R., Fujioka, H., Williams, P. S., Chalmers, J. J., Grimberg, B., Zimmerman, P. A. and Zborowski, M., The FASEB journal, 2006, 20, 747-749. Sullivan, D. J., Biopolymers Online, 2005. Pries, A., Secomb, T. and Gaehtgens, P., Cardiovascular research, 1996, 32, 654-667. Herricks, T., Antia, M. and Rathod, P. K., Cellular microbiology, 2009, 11, 1340-1353. Shelby, J. P., White, J., Ganesan, K., Rathod, P. K. and Chiu, D. T., Proceedings of the National Academy of Sciences, 2003, 100, 14618-14622. Adams, J. D., Kim, U. and Soh, H. T., Proceedings of the National Academy of Sciences, 2008, 105, 18165-18170. Lee, H., Jung, J., Han, S.-I. and Han, K.-H., Lab on a Chip, 2010, 10, 2764-2770. Plouffe, B. D., Mahalanabis, M., Lewis, L. H., Klapperich, C. M. and Murthy, S. K., Analytical chemistry, 2012, 84, 1336-1344. Forbes, T. P. and Forry, S. P., Lab on a chip, 2012, 12, 1471-1479. Bu, M., Christensen, T. B., Smistrup, K., Wolff, A. and Hansen, M. F., Sensors and Actuators A: Physical, 2008, 145, 430-436. Han, K. H. and Bruno Frazier, A., Journal of Applied Physics, 2004, 96, 5797-5802. Han, K. H. and Frazier, A. B., Microelectromechanical Systems, Journal of, 2005, 14, 1422-1431. Han, K. H. and Frazier, A. B., Lab Chip, 2006, 6, 265-273. Paul, F., Melville, D. and Roath, S., Magnetics, IEEE Transactions on, 1982, 18, 792-795. Trager, W. and Jensen, J. B., Science (New York, NY), 1976, 193, 673. Lambros, C. and Vanderberg, J. P., The Journal of Parasitology, 1979, 418-420. Owen, C., Biophysical journal, 1978, 22, 171-178.


23


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(40) (41) (42) (43) (44) (45) (46)

Page 24 of 24

Qu, B. Y., Wu, Z. Y., Fang, F., Bai, Z. M., Yang, D. Z. and Xu, S. K., Analytical and bioanalytical chemistry, 2008, 392, 1317-1324. Melville, D., Paul, F. and Roath, S., Magnetics, IEEE Transactions on, 1975, 11, 17011704. Takayasu, M., Duske, N., Ash, S. and Friedlaender, F., Magnetics, IEEE Transactions on, 1982, 18, 1520-1522. Hackett, S., Hamzah, J., Davis, T. and St Pierre, T., Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 2009, 1792, 93-99. Zborowski, M., Ostera, G. R., Moore, L. R., Milliron, S., Chalmers, J. J. and Schechter, A. N., Biophysical journal, 2003, 84, 2638-2645. Green, J. V., Radisic, M. and Murthy, S. K., Analytical chemistry, 2009, 81, 9178-9182. Lee, G.-B., Chang, C.-C., Huang, S.-B. and Yang, R.-J., Journal of Micromechanics and Microengineering, 2006, 16, 1024.

(47)

Nam J., Lim H., Kim D., Shin S., Separation of platelets from whole blood using standing surface acoustic waves in a microchannel. Lab Chip, 2011, 11, 3361.

(48)

Nam J., Lim H., Kim D., Jung H. Shin S., Continuous separation of microparticles in a microfluidic channel via the elasto-inertial effect of non-Newtonian fluid, Lab Chip, 2012, 12, 1347.

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Magnetic Separation of Malaria-Infected Red Blood Cells in Various

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