Malaria Diagnosis Using Magnetic Nanoparticles

Clement Yuen, Quan Liu* Division of Bioengineering, School of Chemical and Biomedical Engineering,

Nanyang Technological University, Singapore 637457 * Email: quanliu@ntu.edu.sg

Abstract- The current gold standard method of Malaria

diagnosis relies on the blood smears examination. The method is labor-intensive, time consuming and requires the expertise for data interpretation. In contrast, Raman scattering from a metabolic byproduct of the malaria parasite (Hemozoin) shows the possibility of rapid and objective diagnosis of malaria. However, hemozoin concentration is usually extremely low especially at the early stage of malaria infection, rendering weak Raman signal.

In this work, we propose the sensitive detection of enriched �-hematin, whose spectroscopic properties are equivalent to hemozoin, based on surface enhanced Raman spectroscopy (SERS) by using magnetic nanoparticles. A few orders of magnitude enhancement in the Raman signal of �-hematin can be achieved using magnetic nanoparticles. Our result demonstrates the potential of SERS using magnetic nanoparticles in the effective detection of hemozoin for malaria diagnosis.

I. INTRODUCTION

Human malaria is a major disease worldwide, causing 243 million cases and accounting for nearly one million deaths per year [1]. Malaria disease control, including both diagnosis and treatment, becomes an important global health issue. Since effective drugs for malaria treatment have not been developed, malaria diagnosis is important in arena of malaria disease control. In malaria diagnosis, blood smear examination is the current “gold standard”. However, this method is labor-intensive, time consuming and requires special expertise in data interpretation.

Recently, Raman spectroscopy technique was found being

able to detect biocrystals (hemozoin), produced by the malaria parasite [2]. This technique provides a potential alternative for malaria diagnosis. However, Raman signals can be weak in this method due to the extremely low concentration of hemozoin especially in the initial stage of malaria infection [3]. Therefore, we propose to employ magnetic nanoparticles for the surface enhanced Raman spectroscopy (SERS) for fast, sensitive and objective detection of hemozoin. As the first step, we investigate the use of SERS for detecting enriched �-hematin, whose Raman fingerprints are equivalent to hemozoin.

II. MATERIALS AND METHODS

A. Synthesis of �-hematin

The �-hematin crystals were synthesized by using an acid-catalyzed method [2]. In a constant temperature bath of 60°C with constant stirring (~ 150 rpm), Ferriprotoporphyrin IX was added to NaOH of 0.1 M, followed by the introduction of HCl (0.1 M) to neutralize the alkaline mixture within 10 minutes. After another 3 minutes a sodium acetate buffer solution (4.5 M) with a pH of 4.5 was added into the mixture to maintain an appropriate pH for reaction. The mixture was allowed to react for 60 min prior to being washed with deionized water and filtered. The residue from filtration was collected and dried over P2O5 for ~ 48 hr.

B. Synthesis of Fe3O4@Ag nanoparticles A seed-growth reduction method [4] was used to fabricate

nanoparticles with Fe3O4 core and Ag shell (Fe3O4@Ag). Purchased Fe3O4 magnetic nanoparticles (diameter of ~ 50 nm) were mixed with an AgNO3 solution (Ag salt concentration of 1.77 × 10-4 M and shaken for 30 min, prior to the addition of hydroxylamine hydrochloride to reduce Ag salt for another 5 min. An AgNO3 solution with an Ag salt concentration of 1.77 × 10-4 M was added again to the mixture to ensure growth of Ag on Ag seeds formed on each Fe3O4 core during the first AgNO3 addition and allowed to react for 10 min with vigorous shaking. The final mixture was collected by a magnet and washed with deionized water.

C. Raman Instrumentation A 785 nm – laser was used to excite all the samples.

Excitation power of 20 mW and 2 mW, respectively, were employed for the study of �-hematin based on ordinary Raman spectroscopy and SERS.

III. RESULTS Fig. 1 gives the field emission scanning electron

microscope (FESEM) image of fabricated �-hematin. Each �-hematin crystal shows to be elongated with a typical dimension of about 1 um × 200 nm. The crystals have distinguishable facets and show an external appearance resembling hemozoin biocrystals reported in the literature [5].

Fig. 1. FESEM image of fabricated �–hematin.

Fig. 2 illustrates the FESEM image of the Fe3O4@Ag

nanoparticles. The Fe3O4@Ag nanoparticles are circular in shape with a mean diameter of ~ 60 nm, thus, the Ag shell is around 5 nm thick based on a mean diameter of 50 nm for the Fe3O4 core. The Fe3O4@Ag nanoparticles are aggregated together in Fig. 2, since the nanoparticles are magnetic at room temperature.

Fig. 2. FESEM image of Fe3O4@Ag nanoparticles.

Fig. 3 gives (a) SERS Raman spectrum of �-hematin in the

close proximity of the SERS-active Fe3O4@Ag nanoparticles under the influence of a magnetic field and (b) ordinary Raman spectrum of �-hematin. Raman peaks, such as, 264 cm-1, 753 cm-1, 975 cm-1, 1121 cm-1, 1241 cm-1, 1377 cm-1, and 1623 cm-1, can be observed in both the enhanced and ordinary Raman spectra of fabricated �-hematin, showing a degree of crystallinity comparable to that reported in the literature [6,7]. In addition, the SERS spectrum of �-hematin are augmented by a few orders of magnitude compared with the ordinary Raman spectrum of �-hematin at the same concentration. The improvement in the Raman signal can be attributed to the electromagnetic and chemical enhancements between the Fe3O4@Ag nanoparticles and � - hematin in a

close range under the influence of a magnetic field. Therefore, the Fe3O4@Ag nanoparticles are effective in enhancing the Raman signal of fabricated �-hematin crystals.

Fig. 3. (a) SERS spectrum of �-hematin in the close proximity of the

SERS-active Fe3O4@Ag nanoparticles under the influence of a magnetic field. The excitation power was 20 mW. (b) Ordinary Raman spectrum of

�-hematin. The excitation power was 2 mW.

IV. CONCLUSION In conclusion, the magnetic nanoparticles with silver shell

can enhance the Raman signal of �-hematin under the influence of a magnetic field, showing the great potential for use in malaria diagnosis.

ACKNOWLEDGMENT This research was supported by the Bill and Melinda Gates

Foundation (Grant No. OPP1015169).

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