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Malaria pigment crystals as magnetic micro-rotors: key for high-sensitivity diagnosis

A. Butykai,1 A. Orban,1 V. Kocsis,1 D. Szaller,1 S. Bordacs,1 E. Tatrai-Szekeres,1

L.F. Kiss,2 A. Bota,3 B.G. Vertessy,4, 5 T. Zelles,6 and I. Kezsmarki*1

1Department of Physics, Budapest University of Technology and Economics and Condensed

Matter Research Group of the Hungarian Academy of Sciences, H-1111 Budapest, Hungary2Institute for Solid State Physics and Optics, Wigner Research Centre for Physics,

Hungarian Academy of Sciences, H-1525 Budapest, Hungary3Department of Biological Nanochemistry, Institute of Molecular Pharmacology,

Research Center for Natural Sciences, Hungarian Academy of Sciences, H-1025 Budapest, Hungary4Institute of Enzymology, Research Center for Natural Sciences,

Hungarian Academy of Sciences, H-1113 Budapest, Hungary5Department of Applied Biotechnology and Food Science,

Budapest University of Technology and Economics, H-1111 Budapest, Hungary6Department of Oral Biology, Semmelweis University, H-1089 Budapest, Hungary

(Dated: December 12, 2012)

The need to develop new methods for the high-sensitivity diagnosis of malaria has initiated aglobal activity in medical and interdisciplinary sciences. Most of the diverse variety of emergingtechniques are based on research-grade instruments, sophisticated reagent-based assays or rely onexpertise. Here, we suggest an alternative optical methodology with an easy-to-use and cost-effectiveinstrumentation based on unique properties of malaria pigment reported previously and determinedquantitatively in the present study. Malaria pigment, also called hemozoin, is an insoluble mi-crocrystalline form of heme. These crystallites show remarkable magnetic and optical anisotropydistinctly from any other components of blood. As a consequence, they can simultaneously act asmagnetically driven micro-rotors and spinning polarizers in suspensions. These properties can gainimportance not only in malaria diagnosis and therapies, where hemozoin is considered as drug targetor immune modulator, but also in the magnetic manipulation of cells and tissues on the microscopicscale.

In spite of the global efforts made for its elimination in-cluding preventive strategies and drug therapies, malariais still the topmost vector-borne infectious disease withmore than 200 million clinical cases and around 1 mil-lion fatalities a year1. Increasing drug resistance of theparasites strongly acts against the global malaria control,while climate change can even result in the reintroduc-tion of malaria mosquitos into post-endemic countries.A significant improvement could be achieved via the de-velopment of cheap diagnostic methods accurate even atthe early stage of the infection and via new drugs or vac-cines efficient against the most severe types of malariaparasites2,3.

Among diagnostic methods currently in practice themost reliable and sensitive one is the microscopic ob-servation of blood smears –able to detect parasitemiaassociated with 5-10 parasites in 1µl blood–, which israther costly as requiring expertise and high-powered mi-croscopes. Though antigen-based detection of malariaparasites offers a cheaper alternative and the correspond-ing rapid diagnostic tests (RDT) are widely used4–6,presently these techniques have strong limitations. Per-haps the two major drawbacks are that i) RDTs are notsensitive enough to detect early-stage infections, the cur-rent sensitivity threshold being around 100parasites/µland ii) the tests are not quantitative enough to distin-guish between levels of infections (in endemic areas, mostindividuals will test positive showing some degree of par-asitemia but not allowing identification of patients withactive disease requiring urgent treatment)7. Addition-

ally, false positive results may arise due to the imperfectclearage of antigen proteins from the body after success-ful treatments, while false negative results are owing totheir absence in certain Plasmodium strains8, as is thecase for histidine-rich protein II9. Although among themolecular biology-based methods, polymerase chain re-action (PCR) assays are sensitive enough to detect 1 par-asite /µl10,11, the practical use of PCR assays on the fieldis limited due to requirements of sophisticated technologyand expertise.

In the last few years, the need to develop new diag-nostic methods has been driving extended research anda large arsenal of diagnostic schemes has been proposed.Some of them are still based on selective microscopic de-tection of infected blood cells such as magnetic deposi-tion microscopy12, third harmonic generation imaging13,fluorescent study of cell microarray chips14 and photoa-coustic flowmetry15. There is an increasing number ofmethods using malaria pigment as the target materialfor magnetic diagnosis including magnetic purificationprocesses12,16, magneto-optical detection using polarizedlight17, electrochemical magneto immunosensors19 andmagnetic field enriched surface enhanced resonance Ra-man spectroscopy20.

Malaria pigment, also called hemozoin, is a byproductof the disease formed during the intraerythrocytic growthcycle of the parasites21,22. Digestion of hemoglobinby the malaria parasites results in the accumulation ofmonomeric heme. As it is highly toxic to the par-asites, they transform heme into an insoluble crystal-

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2

lized form in which heme groups are dimerized throughironcarboxylate links and the three dimensional struc-ture is stabilized via hydrogen bonds23. This processis accompanied by the change in the valency and thelocal coordination of iron and leads to the transforma-tion of low-spin diamagnetic Fe2+ ions contained in oxy-hemoglobin into high-spin (S=5/2) paramagnetic Fe3+

ions in hemozoin24–26. Besides playing a key role in sev-eral diagnostic techniques, malaria pigment, an ensembleof submicron-sized paramagnetic hemozoin crystallites,is a main drug target and may also act as an immunemodulator27,28.Hemozoin has a low-symmetry triclinic crystal

structure29 as shown in Fig. 1a. Although the mor-phology of the crystallites shows variations dependingon the parasite species30,31, they typically have an elon-gated rod-like shape with a length ranging from 300 nm to1µm. Besides the natural formation of hemozoin insidethe parasites, various methods have been established forits chemical synthesis23,32. Though the artificially grownversion is usually called β-hematin to be distinguishedfrom hemozoin, they are demonstrated to share identicalchemical composition, crystal structure23,29, optical23,33

and magnetic properties24,25. Hereafter, we will refer toboth natural and synthetic versions of malaria pigment ashemozoin. Hemozoin crystals used in the present studywere prepared from hemin by an aqueous acid-catalyzedreaction28. Transmission electron micrograph (TEM) im-ages of typical crystallites are shown in Fig. 1c. Similarlyto the naturally grown ones, they are elongated and char-acterized by a size distribution of ∼700±200nm.Here, by extending the work of Newman and

coworkers17 we report a new path for the magnetic detec-tion of malaria, which, in contrast to most of the afore-mentioned recently emerging techniques, may be real-ized as a cheap and compact diagnostic tool without theapplication of research-grade instruments. Unique mag-netic and optical properties of malaria pigment crystalsas well as their highly controllable dynamics in fluids,which all play key roles in the principle of detection,are systematically investigated. The threshold of our de-vice detecting hemozoin content, as in the present trialstate, is 15 picogram of hemozoin in 1µl blood equiva-lent to a level of parasitemia .30 parasites/µl17, whichneeds to be confirmed via extended clinical trials. Whilethis detection limit already shows improvement over thatof RDTs, it is further decreased by about more thanone order of magnitude for hemozoin detection in bloodplasma or serum corresponding to a parasitemia less than1 parasite/µl.

Results

The diagnostic methodology presented in this paperrelies to a large extent on magnetic properties which arehighly specific to malaria pigment crystals and unique inhuman body. First we review the fundamental charac-

C4v

a

b

c

O

Fe

N

C

1 !m2 !m

a

b

c

ba

c

FIG. 1: | Structure and morphology of hemozoin crys-

tals. (a) Triclinic structure of hemozoin with two unit cellsdisplayed using structural data from Ref. 29. The main crys-tallographic axes a, b and c are also indicated. (b) The localsymmetry of five-fold coordinated iron in hemozoin nearlypreserves a four-fold rotation axis, C4v . The angle spannedby this C4v axis (hard axis of the magnetization) and thecrystallographic c-axis (fore-axis of the elongated crystals) isδ≈60o, where the c-axis points out of the plane of the figure.(c) Transmission electron micrographs of typical hemozoincrystallites dried from suspensions.

teristics of the crystallites determined by magnetizationand magneto-optical measurements. The following partdescribes the dynamics of their magnetically driven rota-tion in fluids with different viscosity such as hemolyzedblood, water, acetone, etc.

Magnetic anisotropy of hemozoin. The low crys-tal symmetry would generally imply that hemozoin is ahighly anisotropic paramagnet with different magneticsusceptibility values along each of the three main crys-tallographic axes. However, Fe3+ ions located in the cen-ter of porphyrin rings experience higher local symmetrysince the four-fold rotational axis perpendicular to theplane of the porphyrin unit is nearly preserved as shownin Figs. 1a-b. Therefore, we expect that the magneticproperties of malaria pigment, which are mainly deter-mined by Fe3+ ions, reflect this axial (C4v) symmetry

3

FIG. 2: | Magnetic orientation and dynamics of paramagnetic hemozoin crystals with anisotropic easy-plane

character. In these schematic drawings, the cylinders represent the suspended hemozoin crystals. The axes of the cylinderscorrespond to the magnetic hard axes of the crystals and not related to their fore-axes. (a) Without external magnetic field thecrystals in the suspension are randomly oriented. (b) With the application of a magnetic field, the hard axes of the crystalsbegin to align perpendicular to the magnetic field vector B, though this orientation is hindered by the thermal fluctuations.(c) In the high-field limit this two-dimensional alignment is completed, with the hard axis of each crystal lying within the planenormal to the field. (d) In slowly rotating fields the crystallites behave as magnetically driven micro-rotors. (e) Due to theviscosity of the fluid, at high rotation frequencies their hard axes tend to align parallel to the rotation axis and consequentlythey stop spinning. Only in this case a full three dimensional alignment of the hard axes is achieved.

and hemozoin behaves either as an easy-axis or as aneasy-plane paramagnet.Based on a multi-frequency high-field electron para-

magnetic resonance (EPR) study on powder samples,Sienkiewitz and co-workers25 suggested that the be-haviour of the S=5/2 spins of Fe3+ ions in hemozoincan be described by the following Hamiltonian, H =

D(

S2z − S(S+1)

3

)

+ E(S2x − S2

y) + µBgBS, where D in

the first term is the zero-field splitting associated with anaxial anisotropy, while lowering of the C4v symmetry in-troduced via E is negligible as |E/D| ≤ 0.035. They alsofound that the Zeemann-splitting induced by an externalmagnetic field, B, is characterized by a nearly isotropic g-factor, g≈2. The dominance of the D term together withits positive sign found in this low-temperature EPR study– in agreement with the results obtained by Mossbauerspectroscopy24 – hint toward the fact that hemozoin canbehave as an easy-plane paramagnet over an extendedtemperature region. According to the local symmetrygenerated by the ligand field at iron sites, we assign theeasy plane of the magnetization with the plane of theporphyrin rings, hence, the hard direction labeled as z-axis in the spin Hamiltonian above coincides with thefour-fold rotational axis.When such easy-plane crystallites suspended in a liq-

uid are exposed to an external magnetic field, they tendto co-align with the field direction to gain magnetic en-ergy (see Figs. 2a-c). Assuming a linear field dependenceof the magnetization, which is experimentally confirmedat room temperature, the magnetic anisotropy energy

is U=− 12B2

µ0

cos2(θ)(χzz − χxx)V . Here, χzz and χxx

stand for the linear magnetic susceptibility of a crys-tal along the hard axis and within the easy plane, re-

spectively, θ is the angle between the direction of thefield and the hard axis of a crystal and V is its volume.Magnetization densities for fields applied within the easyplane and along the hard axis of the crystal are given byMx = χxxB/µ0 and Mz = χzzB/µ0, respectively. (Fordetails see Methods section.) Since thermal fluctuationstry to restore the random orientation, the angular distri-bution of the crystals over the suspension depends on therelative strength of the magnetic anisotropy energy andthe energy scale of thermal fluctuations, kBT , according

to f(θ) = e−U/kBT

2π∫

π0

e−U/kBT sin θdθ.

In order to directly determine the strength of the mag-netic anisotropy we car ried out field- and temperature-dependent magnetization measurements on powder sam-ples of randomly oriented crystals as well as on crystalssuspended in a mixture of 70% water and 30% glycerol.In the second case, the measurements were performedboth after zero-field cooling for maintaining the randomorientation of the crystals in the suspension and after afield cooling process used to magnetically align the crys-tals and fix them by freezing the mixture. (Fixation oc-curs below the freezing point of the mixture, Tfr≈230K– see Supplementary Information.) For suspensions withhemozoin content less than 10µg/µl, the magnetic fieldof B=5T used for field cooling from room temperaturedown to T=2K was found safely large to achieve highdegree of orientation within the suspensions as schemat-ically illustrated in Fig. 2c. This way we could measurethe magnetization specific to the case when the magneticfield lies within the easy plane and the hard axis of eachcrystal is aligned perpendicular to the field.

Here, we note that the magnetic hard axis is roughlyparallel to the [131] crystallographic axis, while the

4

0 1 2 3 4 5 6 7 8 90

1

2

3

4

5

S=5/2 Brillouin Mx (aligned) Mx (calculated) <M> (powder) <M> (random) <M> (calculated) Mz (calculated)

M [

B/F

e]

B [T]

T=2 Ka

0.00 0.04 0.08 0.12 0.16 0.200.0

0.2

0.4

0.6

0.8

270 275 280 285 290 295 300

0.012

0.014

0.016

5 K bB=0.5 T

M [

B/F

e]

1/T [K-1]

300 K

M [

B/Fe]

T [K]

FIG. 3: | Magnetization anisotropy of malaria pig-

ment crystals. (a) Field dependence of the magnetiza-tion measured at T=2K for a powder sample, randomly ori-ented (zero-field cooled suspension) crystals and magneticallyaligned (field cooled suspension) crystals are shown by blueopen circles, blue dots and green dots, respectively. As ex-pected, the former two are essentially identical. Magnetiza-tion curves calculated for fields lying within the easy planeand pointing along the hard axis of a crystal are also plottedwith green and red lines, respectively. The angular average ofthe magnetization corresponding to the random orientationof the crystals is also displayed with blue line. (For details ofthe calculation see the Methods section.) Magnetization val-ues are given for a single iron site in Bohr-magneton units. Toemphasize the anisotropic character of hemozoin, Brillouin’sfunction describing the magnetization of an isotropic S=5/2spin is also shown (dashed grey line). (b) Low-field magneti-zation of hemozoin as a function of the inverse temperaturemeasured in B=0.5T. The position of 300K and 5K are indi-cated on the upper scale. The inset shows the data on a lineartemperature scale around 300K. Symbols and lines indicaterespectively the same measured and calculated quantities asin panel (a).

crystallites are elongated along the [001] direction34,35.Therefore, the two dimensional co-alignment of the hardaxes of the crystals (shown in Fig. 2c) leaves a large free-dom for the orientation of their fore-axis since the crystalscan rotate around both their hard axes and the direc-tion of the external magnetic field without any change

in their magnetic energy. This is in agreement withour TEM observations (see Supplementary Information),which showed that magnetic alignment is not straightfor-wardly manifested in the orientation of the shape of thecrystals, in contrast to former assumptions17.

The field dependence of the magnetization at T=2Kand the temperature-dependent magnetization measuredin B=0.5T with increasing temperature are plotted inFig. 3a and 3b, respectively, for powder samples, zero-field cooled and field cooled suspensions. The differencebetween the magnetization of the randomly oriented andthe aligned samples increases with decreasing tempera-ture and clearly shows the anisotropic nature of hemo-zoin.

To determine the magnetization also for fields pointingalong the hard axis of a given crystal from these data, wecalculated the magnetization, both as a function of fieldand temperature, using the spin Hamiltonian with axialanisotropy and tuned the value of D to obtain the bestfitting with the measured curves. We found that themagnetization of a crystal strongly depends on the angleθ spanned by its hard axis and the direction of the exter-nal field. Besides the magnetization density values corre-sponding to the easy plane (Mx) and the hard axis (Mz),the magnetization of a sample containing randomly ori-ented crystals, 〈M〉, was also evaluated by averaging overθ. All the experimental data shown in Fig. 3 and addi-tional data presented in the Supplementary Informationare well reproduced by a common value of the single fit-ting parameter, D=13.4K, which is consistent with thevalue reported in former EPR25 and Mossbauer24 spec-troscopic studies.

For the anisotropy of the low-field magnetization (or al-ternatively the linear susceptibility) we obtained a valueas large as Mx/Mz = 9.6±0.2 at T=2K. Though this ra-tio is gradually reduced when the energy scale of the ther-mal fluctuations becomes comparable and larger than thezero field splitting, i.e. for kBT > D, it is still consider-able at T=300K with Mx/Mz ≈ 1.16±0.03. The magni-tude of the anisotropy at room temperature implies thatpartial (two dimensional) and full (three dimensional)magnetic alignment of the crystals respectively obtainedby static and rotating fields can be achieved by magneticfields of .1T as proposed in Fig. 2.

Magnetically induced linear dichrosim of

malaria pigment. Similarly to the magnetic anisotropy,the planar stacking of Fe3+-protoporphyrin-IX units inhemozoin29 together with the axial symmetry of iron sitesare indicative of anisotropic optical properties for a sin-gle crystal (see Figs. 1a-b). More specifically, optical ex-citations in the absorption spectrum of hemozoin overthe near-infrared and the visible regions can be assignedto transitions mainly involving π and π∗ orbitals of theporphyrin and d orbitals of the central Fe3+ ion36,37.These assignments also support the local C4v symmetryof iron in hemozoin similarly to the case of hemin anddeoxyhemoglobin36. Since the same symmetry dictatesthe magnetic and optical anisotropy of hemozoin on the

5

-400 -300 -200 -100 0 100 200 300 4000.0

0.2

0.4

0.6

0.8

1.0

=475 nm =585 nm =670 nm

T /T

[a.

u.]

B [mT]

0 0.5 1.0 1.5 [ m]

FIG. 4: | Magnetically induced linear dichroism in

hemozoin suspensions. Magnetic field dependence oflinear dichroism measured on a room-temperature aqueoussuspension of hemozoin at wavelengths λ =475 nm (bluesquares), 585 nm (orange triangles) and 670 nm (red dots).Data corresponding to different wavelengths were normalizedto a common scale, which resulted in a universal field depen-dence reproduced well by the theory. Assuming an average-sized crystal, the fitting (dotted line) yields Mx/Mz=1.11 forthe magnetization anisotropy, which corresponds to Mx −Mz=0.013µB/Fe in a magnetic field of 5 T. The quality ofthe fit can be further improved (solid line) by assuming thedistribution of crystal size shown in the caption. For detailssee the main text.

microscopic level, alignment of the crystallites by exter-nal field is expected to simultaneously generate macro-scopic magnetic and optical anisotropy in their suspen-sions.This invokes a diagnostic tool based on magneto-

optical phenomena such as magnetically induced linearbirefringence/dichroism or polarization dependent lightscattering. Though all of these three effects can be rel-evant we will refer to them as magnetically induced lin-ear dichrosim (MLD). Recently, Newman and coworkershave reported a magneto-optical methodology capable ofa sensitive diagnosis of malaria17,18. They found a spe-cific field dependence of MLD38. In order to probe themicroscopic properties of hemozoin we revisited this phe-nomenon and studied its wavelength dependence.We investigated the magnetic field dependence of the

linear dichroism on aqueous suspensions of hemozoinat multiple wavelengths (e.g. λ=475nm, 585nm and670nm) by measuring the transmitted intensity in Voigtconfiguration for light polarizations parallel and perpen-dicular to the applied field using a polarization modula-tion technique. (See Methods section for details.) Theexperimental curves obtained at different wavelengthsfollow a universal field dependence when normalized to acommon scale as shown in Fig. 4. After the quadratic in-crease of MLD at low fields, the signal tends to saturatewith an inflection at an intermediate field B0≈0.1T.The mechanism behind MLD in hemozoin suspensions

is outlined schematically in Figs. 2a-c. In a dilute suspen-sion the crystals are oriented randomly, resulting in anoptically isotropic media since polarization effects fromindividual crystals average to zero. As already discussed,in external magnetic fields the crystals align in a man-ner that the field would preferably lie within their easyplanes. The ordering is opposed by thermal fluctuationsand this competition determines the specific field depen-dence of MLD. The linear dichroism of a single hemozoincrystal is characterized by the difference of its transmis-sion coefficients Tx and Tz corresponding to light po-larizations parallel and perpendicular to the porphyrinplanes, respectively. (These directions were respectivelycalled easy plane and hard axis in the former magneticterminology.) In external magnetic field the contribu-tions from individual crystals produce a macroscopictransmission anisotropy between polarizations paralleland perpendicular to the field direction

∆T

T= c ·

Tx − Tz

Tx + Tz·

∫ π

0

πf(θ)(3 cos2 θ − 1) sin θdθ, (1)

where ∆T and T is the difference and the average ofthe transmitted intensities for the two polarizations, re-spectively. The factor c expresses linear scaling with theconcentration. The wavelength dependence emerges inthe second term through the transmission anisotropy ofindividual crystals, while the integral captures the fielddependence, hereafter referred to as Φ(B), describing thedegree of the magnetic alignment39.The universal field dependence shown in Fig. 4 was

fitted by numerically evaluating Φ(B) for different val-ues of the magnetic anisotropy using a typical crystalsize of V=200×200×700nm3. We found the best fit-ting with Mx/Mz = 1.11± 0.04 in good agreement withthe value obtained from the magnetization study on ori-ented samples at room temperature. As the difference ofthe experimental and the fitted curves are likely due tothe size-distribution of the crystals, we refined the fit as-suming lognormal distribution of the crystal size. To re-duce the number of free parameters, we fixed the averagelength of the crystals and the magnetization anisotropyto the values previously obtained by the single-size-fit, i.e.L=700nm and Mx/Mz = 1.1. Then, we obtained 220 nmfor the standard deviation of the length and 8:2 for theaspect ratio of the crystals. These are both realistic inthe light of scanning electron micrographs (aspect ratioof 7:2 was considered in the single-size-fit) and furtherimproved the quality of the fit.Spectral features of the MLD effect in hemo-

zoin. To gain more insight into the microscopic opticalproperties of hemozoin and trace the spectral range op-timal for diagnosis, we studied MLD effect from the ul-traviolet to the near-infrared region (λ=300-1300nm) inB=0.3T. We also investigated the influence of differentsuspension media, including normal saline, blood plasmaand blood, on the detectability of hemozoin.As shown in Fig. 5 the MLD spectra of hemozoin sus-

pensions in saline and blood plasma are essentially iden-

6

300 400 500 600 700 800 9000.0

0.2

0.4

0.6

0.8

1.0

1.2

400 600 800 1000 12000.0

0.2

0.4

0.6

0.8

1.0 S 32 pg/ lS 320 pg/ lS 3.2 ng/ lP 1 ng/ lB 1 ng/ l

T / T

[%

]

Wavelength [nm]

T / T

[%

]Wavelength [nm]

FIG. 5: | Wavelength dependence of the MLD ef-

fect in hemozoin suspensions. MLD spectra for room-temperature hemozin suspensions in normal saline (S), bloodplasma (P) and full blood (B) normalized to a concentrationof 1 ng/µl. The inset shows the MLD effect over a broaderspectral range in normal saline.

tical and exhibit characteristic peaks distributed mostlyover the visible range, which may serve as optical finger-prints of malaria pigment. These peaks are likely dom-inated by the linear dichroism of absorption bands ob-served over the same range32,37. The magnitude of MLDspectra shows linear dependence on the hemozoin con-tent over a wide range of concentrations reassuring thefeasibility of a quantitative diagnosis. The effect is thelargest at λ≈670 nm, where the macroscopic transmis-sion anisotropy reaches ∆T /T =1% for the suspensionwith 1 ng/µl hemozoin content corresponding to an op-tical path of d=10mm. If we assume that only the ab-sorption of the crystals contribute to MLD, i.e. polar-ization dependent light scattering can be neglected, thisvalue corresponds to a robust transmission anisotropyTx−Tz

Tx+Tz≈40% for a typical crystal and a large difference in

its absorption coefficients ∆α=αz − αx≈1.5·104 cm−1.

The MLD effect decreases in blood by a factor of ∼3.The overall sensitivity is further reduced owing to strongabsorption and light scattering by the blood components,mainly by red blood cells. For wavelengths shorter than∼620nm the transmitted intensity drastically drops al-lowing no further observation of the MLD signal. Toapproach the sensitivity level achieved for blood plasmaby visible light, we used hemolyzed blood in followingstudies, which helps to strongly reduce light scattering.Please note that the hemolysis of actually infected bloodsamples is also favourable for the diagnosis, since thehemozoin portion still contained within the erythrocytescan be released into the blood plasma, hence becomingeffectively detectable this way. We also work on thedevelopment of simple techniques for the separation ofhemoglobin from hemolyzed blood, while keeping hemo-

0.1 1 10 100-140

-120

-100

-80

-60

-40

-20

00.0

0.2

0.4

0.6

0.8

1.0

1.2

b

propanol =1.95 mPa·swater =0.89 mPa·smethanol =0.54 mPa·sacetone =0.31 mPa·s

MLD

pha

se [d

egre

e]

f [Hz]

1 ng/ l

a

1 ng/ l

MLD

am

plitu

de [%

]

FIG. 6: | Magnetically driven dynamics of hemozoin

crystals in various suspension media at room tem-

perature. (a)/(b) Semi-logarithmic plot of MLD ampli-tude/phase versus the frequency of the field rotation, f , forhemozoin suspended in propanol, water, methanol and ace-tone with 1 ng/µl concentration. Results in hemolyzed bloodare essentially identical with those obtained in water and notshown here. Viscosity (η) for the different media are alsoindicated.

zoin within the plasma, and on the selective filtering ofhemozoin40.

Malaria pigment crystals as magnetic micro-

rotors. For a sensitive detection of weak polarizationeffects generated by low amounts of hemozoin in infectedblood, it is inevitable to use polarization modulationas was already proposed by Newman and coworkers17

and also applied in our magneto-optical experiments.However, besides polarization modulation of the prob-ing light, – in the special case of hemozoin crystals – alsomagnetic modulation of light polarization is conceivable.

The central idea is that the magnetically aligned crys-tallites follow the direction of a rotating magnetic field,i.e. they behave as magnetically driven micro-rotors ina suspension. Moreover, the dichroic planes of the crys-tals rotate in a synchronous manner, thus the suspensionacts as a spinning polarizer modulating the intensity andthe polarization of the transmitted light beam. The ap-plication of a polarizing beam splitter after the sampleand the differential detection of the two orthogonally po-

7

larized beams by a balanced photodiode-bridge providean efficient scheme for the reduction of intensity noise,meaning that an ordinary laser diode is sufficient as lightsource. From the differential signal the a.c. componentcorresponding to the second harmonic of the rotationfrequency is selectively detected, which originates solelyfrom MLD caused by the rotation of the dichroic crystalsand it is not affected by parasitic intensity noise comingfrom optical, mechanical and thermal instability of thedevice.

A special arrangement of permanent magnets in aring-shaped structure surrounding the sample, calledHalbach-cylinder41, is used to generate a uniform mag-netic field of B=1T at the sample position. The ringis rotated by a d.c. electric motor with a frequency ad-justable over the range of f=0.1-130Hz, resulting in afield which rotates within the plane perpendicular to thelight path. We found that the efficient co-alignment ofthe crystals using the strong and nearly homogeneousmagnetic field of the Halbach-cylinder rotated with fairlylarge frequencies plays crucial role in the sensitivity of ourmethod and leads significant improvements over previousmagneto-optical detection schemes17,18. (For further de-tails on the device see Methods section).

Applying this new methodology, we carried out aphase-sensitive detection of MLD on hemozoin suspen-sions. Besides the amplitude of the signal, its time delayrelative to the rotating field has also been recorded. Tounderstand the dynamics of the crystals, we measuredthe frequency dependence of MLD over f=0.1-130Hz insolvents with different viscosity (see Fig. 6). The ampli-tude of MLD remains constant at low frequencies, thendecays drastically towards higher frequencies. The phaseshift of MLD grows gradually with increasing frequencywith a viscosity dependence exposed more in the high-frequency region.

These results can be understood via the basic featuresof the highly complex crystal dynamics as schematicallyshown in Figs. 2c-e. In a strong static field, as in Fig. 2c,the hard axes of the crystallites are distributed uniformlyin the plane perpendicular to the field direction. Uponthe rotation of this field, the resulting torque forces thehard axes of the crystals to precess around the rotationaxis and follow the field. Due to the viscosity of the fluidthe system behaves as an ensemble of damped rotators.Consequently, towards higher frequencies the crystals ex-perience an increasing angular delay relative to the fieldand their hard axes tend to align parallel to the rota-tion axis. This manifests in the finite phase and thedecreasing amplitude of MLD, respectively. When thisalignment is completed, no magnetic torque acts on thecrystals as the magnetic field rotates within their easyplanes, hence they stop moving. The analysis of rota-tional dynamics for easy-axis and easy-plane magneticparticles has recently been subject to extensive theoret-ical and experimental research42–44. The dynamics de-scribed here is specific to crystals with easy-plane mag-netic anisotropy as was also reported for other easy-plane

paramagnetic particles42,44,45 and fundamentally differsfrom the motion of easy-axis crystallites. In the presenteasy-plane situation, MLD signal is suppressed towardshigh frequencies because optically isotropic planes of thedynamically co-aligned crystals are exposed to the lightand consequently no dichroism can emerge.We tested the concentration threshold of hemozoin

detection achievable by this method. Frequency de-pendence of MLD was measured for aqueous suspen-sions of hemozoin prepared over six orders of magni-tude in concentration, namely from 30ng/µl to 0.5 pg/µl.MLD curves displayed in Figs. 7a-b show that the lowestconcentration corresponding to a parasitemia less than1 parasite/µl is still readily detectable. More relevantto diagnosis, our current detection limit for hemozoin inblood is c=15pg/µl as demonstrated in Figs. 7c-d. Thisexceeds the performance of RDTs and approaches thedetection limit achievable by microscopic observation ofinfected blood. In order to sufficiently reduce the stronglight scattering of red blood cells over the visible range,the experiments were carried out in hemolyzed blood (seeMethods section). Please note, that the concentrationvalues in Figs. 7c-d correspond to hemozoin contents infull blood and not in hemolyzed blood obtained by 20-fold dilution with distilled water. Thus, we can concludethat the sensitivity of the detection in hemolyzed bloodis close to that in water. The precision of the hemozoincontent for the series of blood samples was checked bythe parallel measurement of MLD signal on water withthe same hemozoin concentrations. At the moment thethreshold of our detection is not limited by the signal-to-noise ratio of MLD but by a mainly frequency inde-pendent baseline superimposed on the signal. This weakcontribution to the second-harmonic signal may come e.g.from the Voigt effect of the medium. The reproducibilityof the lowest-concentration data together with the base-line measured for blood sample containing no hemozoinare displayed in the inset of Figs. 7c.

Discussion

By combining magnetization measurements, broad-band magneto-optical spectroscopy and electron trans-mission microscopy we determined quantitatively themagnetic and optical anisotropy characteristic tosubmicron-sized single crystals of hemozoin. Based onthese results we refined previous models describing themagnetic alignment of the crystals in suspensions17,18.These fundamental properties offer a unique path forthe magnetic manipulation and optical detection of thesecrystallites and are also relevant to the developmentof new drugs blocking hemozoin production. Dielec-tric anisotropy may also play role in the co-alignmentof hemozoin crystallites during their nucleation withinthe digestive vacuoles of the parasites35.We studied the dynamics of the crystals during their

magnetically driven rotation in suspensions with differ-

8

0.0

0.3

0.6

0.9

1.2

1.5

1.8

0 2 4 6 8 10 12 14 16

-40

-20

0

0.0

0.3

0.6

0.9

1.2

1.5

1.8

0 2 4 6 8 10 12 140

1

0 2 4 6 8 10 12 14 16

-40

-20

0

MLD

am

plitu

de [%

]

MLD

am

plitu

de [%

]

30 ng/ l3 ng/ l0.3 ng/ l30 pg/ l3 pg/ l1 pg/ l0.5 pg/ l

f [Hz]

MLD

pha

se [d

eg]b

MLD

pha

se [d

eg]

f [Hz]

a

d

10 ng/ l 0.9 ng/ l0.15 ng/ l 90 pg/ l 60 pg/ l 30 pg/ l 15 pg/ l

bloodc

MLD

(a.u

.)

f [Hz]

water

FIG. 7: | Sensitivity of the optical diagnostic method based on the magnetic rotation of hemozoin crystals in

water and blood. (a)/(b) MLD amplitude/phase for hemozoin in water over a limited frequency range optimal in sense ofsignal to noise ratio. (c)/(d) MLD amplitude/phase for hemozoin in blood over the same frequency range. The concentrationof hemozoin varies over five and three orders of magnitude in water and blood, respectively. The amplitude of the MLD signalis normalized to 1 ng/µl hemozoin content. Concentrations of blood samples refer to the hemozoin contents in full blood andnot in hemolyzed blood. The concentration levels of 0.5 pg/µl and 15 pg/µl are still readily detectable in water and blood,respectively. Inset in panel (c) shows the reproducibility for the baseline (black curves) and the lowest-concentration data (withcolor coding used in the main panel).

ent viscosity including hemolyzed blood. Based on thefact that the synchronous motion of such micro-rotorsinduces intensity modulation of the transmitted light viapolarization effects, we assembled a device for the detec-tion of malaria pigment in blood. The device providesa more sensitive way of diagnosis RDTs and approachesthe sensitivity achievable by microscopic observation ofinfected red blood cells, which is the most effective diag-nosis in practice to date. We found that the sensitivityof the hemozoin detection in blood plasma is even higherand we are aiming at the improvement of the detectionthreshold by filtration techniques.

We expect no major reduction of sensitivity for in-fected blood samples compared to the threshold reportedhere for synthetic hemozoin in blood for the following rea-sons. First, hemolysis of infected blood helps to releasethe portion of hemozoin still contained within the ery-throcytes into the blood plasma. Furthermore, our pre-liminary data indicate that the magnitude of MLD signal– observed in large fields (B&1T) rotating with low fre-quencies – shows only moderate changes with crystal sizeand morphology in agreement with previous results17.On the other hand, the frequency dependence of both theamplitude and phase of MLD may provide informationspecific to the type of the parasites as increasing crys-tal size results in a shift of the decay frequency towardshigher values. Nevertheless, both the detection thresholdof our device and its specificity to different parasites need

to be proved via extensive clinical tests.

The use of hemozoin as the marker compound for de-tecting Plasmodium infections has clear advantages overthe currently used RDTs. The production of hemozoinis a defense reaction on behalf of the parasite that trans-forms a host protein into a magnetically detectable com-pound with physico-chemical properties invariable upongenetic variations of the parasites. Therefore, efficiencyof our method is not affected by high rates of their ge-netic variation. In contrast, performance of the antigen-based diagnostics relies on the antigen-antibody reactionwhich may be perturbed upon mutations in the antigen.From this respect, it is important to emphasize that Plas-modium strains are known to show great variability oftheir proteins46,47, thereby potentially jeopardizing theefficiency of recognition by a highly specific antibody de-veloped for a rapid diagnostic test.

Beyond the scope of malaria research and diagnosis,our results can contribute to biomedical applicationsof optically and/or magnetically anisotropic submicron-sized particles48–52. We believe that the magnetic micro-rotor concept recognized specifically for malaria pigmentcrystals can be generally applied for the magnetic control,manipulation and detection of submicron-sized magneticparticles functionalized to interact with biomolecules andcells as it has already been demonstrated e.g. in the studyof the elastic properties of single DNA molecules usingmagnetic beads53.

9

Recently, various applications of rotating magneticfield has also been proposed in material sciences. Theseinclude the three dimensional alignment of magnet-ically anisotropic micro-particles aiming to producemagnetically oriented microcrystal arrays for X-raycrystallography54 or to reinforce composites by super-paramagnetic platelets45. Rotating magnetic fields havealso been applied for investigating the formation and ro-tational dynamics for chains of paramagnetic beads55–57.Our method enables the rotation of strong magnetic fields– also characterized by a high level of homogeneity overa large sample volume – at frequencies ranging from 0.1-130Hz, while the polarization detection scheme supportsmonitoring of the dynamics even when magnetic parti-cles are present at ppm concentrations in solution. Thus,this methodology can help to improve the performancein the applications mentioned above.

Methods

Preparation and characterization. Hemozoincrystals were synthetized following the aqueous acid-catalyzed method described by M. Jamarillo and co-workers28. Hemin was dissolved in NaOH with the drop-wise addition of propionic acid adjusting a pH valueof approximately 4. After annealing the mixture for18 hours at 70 oC, the crystals were separated andwashed with NaHCO3, MilliQ water and MeOH, mul-tiple times, alternately – as prescribed by the authors.According to our TEM measurements the typical size ofthe crystals obtained by this method is approximately200×200×700nm3.The transmission electron micrographs were obtained

by using two different methods for the fixation of crys-tals. To avoid aggregation of the crystals, in both casesthe suspensions were prepared with hemozoin contents< 10µg/µl and long-term ultrasonication. The aqueoussuspension of hemozoin crystals was dropped onto for-mvar membrane (purchased from Sigma-Aldrich) placedon 200 mesh copper grid and dried at room tempera-ture. The other method applied for magnetically alignedensembles of crystals was freeze-fracture. In this casethe suspension medium was a mixture of 70% water and30% glycerol to prevent the formation of ice crystals.The droplets (1-2µl) of suspension were pipetted on agold specimen holder kept in a field of B=0.5T at roomtemperature for 30 s, then plunged into partially solidi-fied Freon for 20 s freezing and then placed and stored inliquid nitrogen. Fracturing was carried out at 173K ina Balzers Freeze-fracture Device (Balzers BAF 400 D).The fractured faces were etched for 30 s at 173K. Thereplicas, prepared by platinum-carbon shadowing, werecleaned and washed with distilled water. The membranesand replicas obtained by the two methods were examinedin a transmission electron microscope as shown in Fig. 1and Fig. S1, respectively.For experiments performed on hemozoin crystals sus-

pended in blood plasma and hemolyzed blood, the bloodplasma was obtained via the centrifugation of blood sam-ples and hemolysis of blood was achieved by 20-fold dilu-tion of blood with distilled water. Freshly drawn bloodwas acquired from healthy volunteers.

Magnetization measurements. Magnetizationmeasurements on suspensions were performed using aSuperconducting Quantum Interference Device (SQUID)in magnetic fields ranging from B=0 to 5T at tempera-tures T=2-300K. Powder samples were measured over abroader field range B=0-9T using a magnetometer witha.c. pick-up coil. The magnetization component par-allel to the applied magnetic field was detected. Liquidsamples were placed in hermetically closed plastic straws,while gelatine capsules were used in the case of the solidpowder samples. The diamagnetic baselines originatingfrom the sample holders and the suspension medium weremeasured separately and subtracted from the data.

Magneto-optical methodology. The spectrometercapable of the measurement of MLD over the wavelengthrange of λ=180-1300nm was assembled using a triplegrating monochromator, broad-band light sources (Xe-arc and tungsten lamps) together with a photomultiplierand an InGaAs photodiode as detectors. The experi-ment was set up in Voigt configuration, that is the mag-netic field was applied perpendicularly to the direction ofthe light propagation. Light beam after the sample wascollected using lenses with typical numerical aperture ofNA=0.1-0.2. The fast switching between the light polar-izations parallel and perpendicular to the magnetic fieldwas carried out with a fused silica photoelastic modu-lator operating at a frequency of 50 kHz58. In order toeliminate linear polarization effects other than MLD, thezero-field baseline was measured and subtracted from thefinite-field data.

Diagnostic method and instrumentation. Theprinciples of the new technique have been described in themain text. Figure 8 provides a schematic representationof the diagnostic setup.

Derivation of MLD effect for suspensions. Asdiscussed in the main text, the magnetic and opticalproperties of hemozoin crystals are characterized by anaxial anisotropy. Hence, the value of the complex trans-mission coefficient for light polarization parallel to theC4v axis (z-axis) of a crystal differs from the values corre-sponding to polarizations within the perpendicular plane(xy-plane), i.e. tz 6=tx=ty. The same difference holds forthe magnetization in external magnetic fields pointingalong the z-axis or lying in the xy-plane asMz 6=Mx=My.

While the matrix of the transmission coefficients, t, isdiagonal for each crystal in its own xyz frame, the trans-mission of the whole suspension needs to be calculatedin a common x′y′z′ reference frame, where y′- and z′-axis are conveniently chosen as the direction of the lightpropagation and the magnetic field, respectively. Thenew form of the transmission matrix, t′ – for a crystalwith its z-axis pointing in the direction defined by theazimuth angle, θ and polar angle, φ in the x′y′z′ frame

10

FIG. 8: | Flowchart of the diagnostic setup. The beamfrom the laser diode (1) passes through a polarizer (2) and be-comes vertically polarized. Then it goes through the sampleholder (4) located in the bore of the Halbach magnet (3). Themagnet is rotated with a frequency f by a d.c. motor, thus,the uniform magnetic field of B≈1T at the sample positionrotates within the plane perpendicular to the light propaga-tion. After the sample, the beam is divided into two partswith orthogonal polarizations (±45o) by a Rochon prism (6).The difference and the average of their intensities are detectedby a balanced photodiode bridge (7). The 2f component ofthe difference signal is filtered out by a lock-in amplifier (8)using the reference signal from an optoswitch (5) monitoringthe rotation of the magnet. To obtain the MLD signal, theamplitude of the second harmonic (2f) signal is normalizedwith the average signal by a divisor (9).

– can be obtained by the corresponding base transfor-mation. In this common frame, the portion of the lightintensity transmitted by the crystal for polarization along

the x′/z′ direction is given by Tx′/z′ =∣

∣t′ · ex′/z′∣

∣

2, where

ex′ and ez′ are unit vectors pointing along the x′- andz′-axis, respectively.For an ensemble of the crystals, the macroscopic linear

dichroism exhibited by the suspension is obtained by av-eraging over the contributions from individual crystallitesaccording to ∆T

T= c· Tx−Tz

Tx+Tz·∫ π

0πf(θ)(3 cos2 θ−1) sin θdθ,

where Tx=|tx|2 and Tz=|tz|

2. The distribution of the az-imuth angle is governed by the Boltzmann factor: f(θ) =

e−U/kBT

2π∫ π0

e−U/kBT sin θdθ. The angular distribution is indepen-

dent of the polar angle, since the full rotational symme-try around the magnetic field is preserved. Assuminga linear field dependence of the magnetization, which isexperimentally confirmed at room temperature, the mag-

netic anisotropy energy is U=− 12B2

µ0

cos2(θ)(χzz−χxx)V .

Here, χzz and χxx stand for the linear magnetic suscepti-bility of a crystal along the hard axis and within the easyplane, respectively. The typical volume of a crystal, V ,is approximated as 200×200×700nm3 according to TEMimages. The field dependence of ∆T

Twas evaluated nu-

merically and used for the fitting of the MLD data withtwo free parameters. The first parameter is a scale factor,from which we could obtain the transmission anisotropyof a single crystal, Tx−Tz

Tx+Tz, as a function of the wavelength.

The other parameter is (χzz − χxx)V , which determinesthe distribution of the azimuth angle via the magneticanisotropy energy, hence, it is the only parameter de-scribing the universal field dependence of MLD. Consid-ering an average crystal volume of V=2.8 · 10−20m3, thedifference in the magnetization densities is directly ob-tained.

Numerical calculation of magnetization. Asargued in the main text, the magnetic behaviour ofFe3+ ions with S=5/2 spins in hemozoin is describedby the following axially symmetric Hamiltonian: H =

D(

S2z − S(S+1)

3

)

+ µBgBS. For a given set of {D, B},

we determined the energy eigenvalues (εn) by the nu-merical diagonalization of this 6×6 matrix. Then, themagnetization density vector was obtained according toM = 1

V kBT∂∂B lnZ, where Z is the partition function

in the grand canonical ensemble, Z =(∑

n e−εn/kBT

)N.

Due to the magnetocrystalline anisotropy, the energyeigenvalues depend not only on the strength of the mag-netic field but also on its orientation relative to the crys-tal (θ). For comparison with the experimental data, onlythe component of the magnetization vector parallel tothe field direction was considered.

Besides the principal values Mx and Mz, the mag-netization density of unordered samples were also eval-uated by averaging over the contributions from in-dividual crystals: 〈M〉 = 1

4π

∫ π

0 2π[Mx(Bx) sin θ +Mz(Bz) cos θ] sin θdθ. Mx and 〈M〉 were respectivelymeasured on oriented and random hemozoin suspensionsin a mixture of 70% water and 30% glycerol. In the for-mer case we used a field-cooled freezing procedure. Theexperimental data, both the field and the temperaturedependent magnetization curves, were fitted using thesingle-ion anisotropy factor (D) as the only fitting pa-rameter.

The good correspondence between the values of ax-ial anisotropy found in the present magnetization exper-iments and reported by former EPR25 and Mossbauer24

spectroscopic studies implies that in paramagnetic hemo-zoin crystals magnetocrystalline anisotropy dominatesover the shape anisotropy unlike in usual ferro- and ferri-magnetic crystals with micron or submicron size. This isfurther supported by TEM images recorded from cleavedsurfaces of field-cooled suspensions as presented in theSupplementary Information.

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Acknowledgements We thank G. Mihaly, Y. Tokura,M. Kellermayer and Sz. Osvath for fruitful dis-cussions. This work was supported by Hungar-ian Research Funds OTKA PD75615, CNK80991,CNK81056, Bolyai 00256/08/11, TAMOP-4.2.2.B- 10/1-

2010-0009, TAMOP-4.2.1.B-09/1/KMR-2010-0001, andANR-NKTH ADD-MAL from National Innovation Of-fice.Author Contributions A.B., A.O., L.K., A.Bota,

I.K. performed the measurements; A.B., A.O., S.B., I.K.analysed the data; E. T-Sz. contributed to the samplepreparation; A.B., A.O., V.K., D.Sz., I.K. developed themagneto-optical setup and the diagnostic device; A.B.,A.O., I.K. wrote the manuscript; each author discussedthe results; and I.K. planned and supervised the project.Competing financial interests The authors declare

no competing financial interests.