Dielectrophoresis Trapping and Stretching of Red Blood Cells Infected by Plasmodium Falciparum
Abstraction
We investigated the dielectrophoresis ( DEP ) response of ruddy blood cells ( RBCs ) infected by Plasmodiumfalciparum(Pf-iRBCs ) . In this paper, we present two experimental observations and show how this information can be exploited for favoritism ofPf-iRBCs from clean RBCs and for word picture of RBC mechanical belongingss. The clean RBCs experienced strong positive DEP forces ( 71 pN – 126 pN ) , reflected with a important stretching behaviour at the applied jumping current electromotive force of 3.5 Vrmsat frequences runing from 500 kilohertzs to 5 MHz. In contrast to the extremely stretched clean RBCs,Pf-iRBCs were hardly stretched under assorted electric frequences due to weak DEP effects. This distinguishable behaviour of clean RBCs andPf-iRBCs shows great potency for a label-free separation in combination with shear flow. In add-on, the relationship between DEP force and cell deformability, a quantitative step of cell stretching, agreed with the computational anticipations of cell stretching by optical pincers, proposing DEP method as a promising method for word picture of cell mechanical belongingss.
Keywords: Dielectrophoresis, stretching, deformability, mechanical belongings, ruddy blood cell, malaria
1. Introduction
There has been considerable research attempts in seeking for cellular biomechanical and biophysical belongingss as fresh biomarkers for cell populations and their disease provinces ( 1-4 ) . Assorted preciseness technology attacks have been happening increasing applications in this topic ( 5 ) . These experimental techniques can be coarsely divided into two classs, including sub-cellular and single-cell degrees, such as optical pincers stretching ( 6-8 ) , micropipette aspiration system ( 9 ) , and atomic force microscopy indenture ( 10 ) , and multi-cell degree, such as cell perfusion in microfluidic web ( 11 ) , transit velocity in microfluidic channels with patterned obstructions ( 12, 13 ) , hydrodynamic stretching ( hydropipetting ) ( 14 ) , and dielectrophoresis ( DEP ) stretching of malignant neoplastic disease cells ( 15, 16 ) .
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Malaria is a extremely infective disease of human and a planetary wellness concern. It caused about 207 million instances and more than 627,000 deceases in 2012 ( 17 ) . Plasmodiumfalciparum(P. falciparum) is the deadliest strain of malaria and responsible for 50 % of all malarial infections ( 18 ) . Malaria parasite non merely alters the membrane permeableness of the host RBCs but consumes haemoglobin of host RBCs and signifiers into hemozoin crystals ( 19 ) . Along with this procedure, it causes alterations in electrical belongingss of host cells, which can be sensed by electrical electric resistance ( 20, 21 ) . It was reported that P.falciparuminfected RBCs (Pf-iRBCs ) showed different DEP behaviour than the clean RBCs in two devices designed for sample readying and concentration of parasitized cells from malaria patients ( 22 ) .
Here we present the DEP method non merely for a possible label-free method for separation ofPf-iRBCs at its early phase but besides as a promising method for analysis of mechanical belongingss by pin downing and stretching RBCs. Our experimental observations confirmed the DEP caparison of clean RBCs and weak response ofPf-iRBCs under a wide scope of electric frequences. In combination with numerical simulation, our experimental measurings besides allow a prompt appraisal of mechanical belongingss of clean RBCs.
2. Materials and Method
2.1 P. falciparum Sample
P. falciparumwas cultured in leukocyte-free human RBCs ( Research Blood Components, Brighton, MA ) as antecedently described ( 20 ) . The civilizedP. falciparumsamples were cooled down to room temperature and synchronized at the ring phase. The samples were so washed with phosphate-buffered saline ( PBS ; 2.67 mmol/l KCl, 1.47 mmol/l KH2Polonium4, 137.93 mmol/l NaCl, 8.06 mmol/l Na2HPO4.7H2O ) at 2000 revolutions per minute for 3 proceedingss at 21a?°C and resuspended in an isosmotic buffer ( 9.25 % saccharose with electrical conduction adjusted to 0.055 S/m utilizing PBS ) for DEP analysis. The concluding concentration of RBCs was about 1 % haematocrit.Pf-iRBCs were confirmed utilizing a blue-fluorescent Hoechst Stain solution ( Sigma-Aldrich ) .
2.2 DEP Method
DEP refers to the force exerted on the induced dipole minute of a atom by a non-uniform electric field. The DEP behaviour of a atom is determined by the applied electric field and the Clausius-Mossotti ( C-M ) factor expressed by
where inferiorsPandmbase for atom and medium, severally,
with
being the angular field frequence,
, ? and ? being the permittivity and conduction of the insulator. The clip averaged DEP force is expressed by ( 23 )
whereRis the radius of a spherical atom,
is the permittivity of the medium,
is the existent portion and
is the root mean square value of the electric field.
scopes from –0.5 to 1. When the C-M factor is positive, the polarized atom is traveling toward the electric field upper limit, called positive DEP ( p-DEP ) ; when the CM factor is negative, the atom is traveling off from the electric field upper limit, called negative DEP ( n-DEP ) , as demonstrated in Figures 1 A and 1 C.
2.3 Microfluidic Device and Experiment
The microfluidic device for DEP proving ( Figure 1 B ) consists of an interdigitated electrode construction and a microchamber of 50 µm deep. The Ti/Au electrode of 10 nm/100 nm thickness was deposited on thin glass substrate ( 700 µm ) utilizing E-beam vaporisation and lift-off procedure. The microfluidic channel was fabricated utilizing poly ( dimethylsiloxane ) PDMS casting protocols and bonded to the glass substrate. Alternating current ( AC ) electromotive force of 3.5 Vrmsat frequence from 500 kilohertzs to 50 MHz was applied to the electrode utilizing a map generator. DEP testing ofPf-iRBC samples was performed at room temperature and in a inactive status, observed with an Olympus IX 71 inverted microscope utilizing a 100W HBO lamp.
2.4 Image Analysis and Data Extraction
ImageJ was used to analyse the jutting geometries and dimensions of RBCs. The stretched RBCs were fitted with eclipsiss for quantification with premise of symmetric constellation in cells. COMSOL Multiphysics was used to cipher
in the microfluidic DEP device. A custom book in Matlab R2009a was used to cipher the C-M factor profiles of clean RBCs andPf-iRBCs. All informations are represented as average ± SD unless specified.
3. Consequences and Discussion
3.1 Distinct DEP Behavior of Pf-iRBCs from Uninfected RBCs
As typical cells, such as RBCs are composed of several constituents, including cell membrane and inside. In order to cipher the C-M factor of RBCs surrounded by a specific medium, a smeared-out sphere method was used to come close the effectual permittivity of an N-shell domain as described in literature ( 24 ) . The N-shelled domain can be reduced to an tantamount smeared-out sphere holding an effectual complex permittivity
where
, and
and
are the complex permittivity and radius of the shell N. The effectual complex permittivity of cell was so used to cipher existent portion of C-M factor utilizing Eq. ( 1 ) based on the parametric quantities listed in Table 1.
We observed distinguishable DEP behaviour of clean RBCs fromPf-iRBCs. Figure 2 Angstrom showed the C-M factor profile of unifected RBCs andPf-iRBCs with parasite sizes of 1 µm to 3 µm for a rough estimate of the early phase. It can be seen that the C-M factor of clean RBCs is from 0.25 to 0.6 under electric frequences from 500 kilohertzs to 5 MHz, stand foring the strong p-DEP consequence. In contrast, C-M factor ofPf-iRBCs was around zero under such frequence scope, proposing an highly weak DEP consequence. Experimental observations confirmed this anticipation, where clean RBCs were trapped at the electrode borders, which agrees with a old study ( 22 ) .
It should be noted that beyond the p-DEP induced caparison, we observed the important stretching of clean RBCs as these cells are deformable. As shown in Figure 2 B, the clean RBCs were extremely stretched while thePf-iRBCs were hardly stretched, as highlighted with dotted circles. Confirmation ofPf-iRBCs were performed in a separate DEP proving with blue-fluorescent Hoechst Stain solution ( Sigma-Aldrich ) ( Figure 2 C ) . The fluorescence image ofPf-iRBCs confirmed the integral constellation under AC electric field. Stretching behaviour was quantified by deformability, the ratio between major and minor axes of stretched RBCs fitted by eclipsiss. At an applied electric excitement of 3.5 Vrms5 MHz, the deformability of clean RBCs is significantly different fromPf-iRBCs (P & A ; lt ; 0.001) utilizing a two-sample t-test ( Figure 2 D ) . This information suggests the potency of label-free separation ofPf-iRBCs at early phase from clean RBCs with shear flow.
3.2 Frequency-Dependent Cell Stretching utilizing p-DEP
Cell-stretching utilizing p-DEP is frequency dependent. Figure 3 Angstrom shows the representative microscopic images of pin downing and stretching of clean RBCs at the borders of electrode fingers under different electric frequences. The stretching ratio is strongly dependent on the electric frequence ( Fiure 3 B ) . The deformability of clean RBCs spans from 1.3 to 2.6 under a frequence scope of 500 kilohertzs to 50 MHz. The maximal deformability was achieved when electric frequence was around 3 MHz.
3.3 Word picture of Mechanical Properties
Using the information of cell deformability and DEP force, we were able to qualify the mechanical belongingss of clean RBCs, utilizing informations under frequences of 500 kilohertzs, 1 MHz, 2 MHz, 3MHz, and 5 MHz when clean RBCs showed noticeable deformability. DEP force was calculated utilizing Eq. ( 2 ) . The electric field in the microfluidic device was calculated utilizing COMSOL Multiphysics. The termwas measured at the distance from the terminal of a cell to the border of electrode fingers ( tantamount to the major axes indicated in Figure 2 D ) , which were extracted from image analysis under specific frequence ( Figure 4 A ) . The value of
ranged from
to
. Relative permittivity of medium was assumed to be 80 at room temperature. The deliberate DEP force at assorted frequences were in the scope of 71 pN to 126 pN ( Figure 4 B ) , with positive and negative mistake values calculated based on the lower and higher bounds of distance of cell’s terminal to the border of electrode fingers. Then, deformability of clean RBCs against DEP force ( Figure 4 C ) was compared to the reported simulation informations for RBC stretching by optical pincers ( 25 ) . The trigons represent the deformability from DEP stretching and the dotted curves represent the deformability from computational anticipations. Ref. 1, Ref. 2 and Ref. 3 were based on an initial membrane shear modulus of 5.3, 7.3 and 11.3 µN/m and a higher shear modulus of 13.9, 19.2, and 29.6 µN/m prior to concluding failure. The deformability-force relationship from DEP stretching agrees with the computational anticipations and with better adjustment with higher shear modulus values ( in an enclosure by Ref. 2 and Ref. 3 curves ) . This information suggests DEP stretching as a promising method for word picture of mechanical belongingss of cells.
4. Decisions
We presented the potencies of DEP as an advanced tool for word picture of mechanical belongingss of RBCs beyond its conventional capablenesss in cell separation and sample concentration. We demonstrated a p-DEP based stretching mechanism, which provides potencies for label-free separation of early phase P.falciparumfrom clean RBCs and capablenesss for abstracting extra biomechanics information of cells. Our measurement agreed really good with the deformability-stretching force relationship of RBCs in malaria samples reported in literature. The force magnitude in our DEP system was in a scope of 71 pN – 126 pN for RBCs, which can be farther tuned by modifying the permittivity of medium and electric field strength, based on the polarizability and mechanical deformability of cells of involvement. This p-DEP based stretching mechanism can be combined with a sophisticated numerical theoretical account for abstracting mechanical belongingss of other cell types ( e.g. malignant neoplastic disease ) and tie ining with their disease provinces.
Recognitions
The writers acknowledge support by the National Research Foundation ( Singapore ) through Singapore-MIT Alliance for Research and Technology ( SMART ) Center ( ID IRG ) , and the U. S. National Institutes of Health ( Grant R01 HL094270 ) .
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Figure 1. DEP in microfluidic system. A. Diagram of DEP response of dielectric atoms in non unvarying electric field. B. Schematic of microfluidic device with insert of electrode geometries and dimensions. C. Suspension of RBCs in inactive status when AC electric is off ; trapped RBCs under p-DEP when AC electric field is on ( 3.5 Vrms5 MHz ) .
Figure 2. Differentiation ofPf-iRBCs from clean RBCs. A. C-M factor profiles of clean RBCs and pf-iRBCs utilizing a smeared-out sphere theoretical account. B. Uninfected RBCs are trapped and stretched whilePf-iRBCs retain unstretched. C. Fluorescence images confirmPf-iRBCs undergoing weak DEP ; the insert confirms a Pf-iRBC that is hardly stretched while a clean RBC is extremely stretched. D. Deformability of clean RBCs ( n=25 ) andPf-iRBCs ( n=10 ) . The applied AC electromotive force was 3.5 Vrmsat 5 MHz.
Figure 3. Caparison and stretching of clean RBCs utilizing p-DEP. A. Microscopic images of cell stretching under different electric frequences. B. Deformability of clean RBCs under assorted electric frequences ( n =25 ) .
Figure 4. Word picture of mechanical belongingss of RBCs utilizing DEP stretching. A. Distance of cell’s terminal to the border of electrode fingers. B. Calculated DEP force against electric frequence. C. Relationship of cell deformability and stretching force: comparing of DEP method to the computational anticipations of optical pincers stretching, adapted from mention ( 25 ) . Ref. 1 curve: µ0= 5.3 µN/m, µdegree Fahrenheit= 19.2 µN/m ; Ref. 2 curve: µ0= 7.3 µN/m, µdegree Fahrenheit= 13.9 µN/m ; Ref.3 curve: µ0= 11.3 µN/m, µdegree Fahrenheit= 29.6 µN/m.
Table 1. Parameters utilized to cipher C-M factors of clean RBCs andPf-iRBCs.
|
Cell |
Membrane thickness ( nanometer ) |
Diameter ( µm ) |
Relative permittivity, ?R(22) |
Conductivity, ? ( S/m )(22) |
||
|
Red blood cell |
4.5 |
7 |
Membrane |
4.44±0.45 |
& A ; lt ; 10-6 |
|
|
Interior |
59±6 |
0.31±0.03 |
||||
|
Pf-iRBC |
Host cell |
4.5 |
7 |
Membrane |
9.03±0.82 |
7±2 x 10-5 |
|
Interior |
58±10 |
( 0.95±0.05 ) a?™?m |
||||
|
Parasite |
5 |
1-3 |
Membrane |
8±4 |
& A ; lt ; 10-6 |
|
|
Interior |
70±5 |
1.0±0.4 |
||||
