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Biomedical Optics & Medical Imaging

Ultrasensitive surface-enhanced Raman spectroscopy for malaria diagnosis

Silver nanoparticles are used in two different strategies to achieve high sensitivity to parasites and thus facilitate early malaria diagnosis.
27 July 2016, SPIE Newsroom. DOI: 10.1117/2.1201606.006526

Malaria is a global infectious disease that causes 438,000 deaths a year around the world.1 As the malarial infection can induce death soon after the appearance of the first symptoms, rapid and early diagnosis is an important part of the control and treatment of the disease. At present, microscopic examination of Giemsa-stained blood smears is considered the ‘gold standard’ for malaria diagnosis. This process, however, is time-consuming, and it requires the use of a central laboratory and skilled operators (especially problematic in low-resource regions).

Purchase SPIE Field Guide to MicroscopyTo overcome these problems, several other methods for malaria diagnosis have been developed. These include flow cytometry,2 rapid diagnostic tools,3 the quantitative buffy coat method,4 and the polymerase chain reaction (PCR).5 Although these methods have made a significant contribution to the field of malaria diagnosis, most of them suffer from low sensitivity, high cost, or complicated sample preparation. In addition, the use of Raman spectroscopy has shown great potential for the detection of hemozoin (a unique biomarker of malaria parasites during the early stage of infection).6 The sensitivity levels of such Raman spectroscopy techniques (including conventional surface-enhanced Raman spectroscopy7), however, are insufficient to realize early malaria diagnosis.

In our work,8 we have therefore been developing ways to further enhance the sensitivity of surface-enhanced Raman spectroscopy (SERS) for malaria detection. With our ultrasensitive SERS techniques we can facilitate diagnosis during the early stages of infection (i.e., when the hemozoin level is very low). In our approach, we enhance the Raman signal (by several orders of magnitude) by binding the analyte molecule (hemozoin) to SERS-sensitive nanostructures or nanoparticles (made of noble metals such as silver). The precise enhancement factor depends on the distance between the molecule and the metal surface. In our investigation we have therefore focused on achieving a tight contact between hemozoin and silver nanoparticles to enhance the SERS signal. This is a non-trivial task, however, because hemozoin is a nanocrystal (about 300nm long and 100nm in diameter)9 and its concentration is extremely low during the early stages of the malaria infection.

In our first strategy, we explored using the magnetic property of hemozoin to achieve the tight contact between it and the SERS-sensitive nanoparticles. Hemozoin is paramagnetic and thus exhibits an induced magnetic field when it is in close proximity to an external magnet. We took advantage of this property by coating a SERS-sensitive silver (Ag) layer on the outside of a magnetic iron oxide (Fe3O4) nanoparticle to form a nanoshell structure (Fe3O4@Ag). In this setup, the nanoshell is attracted toward a nearby hemozoin crystal when an external magnet is present. In addition, both the hemozoin crystals and the nanoshells are attracted toward the magnet and so the concentration of both becomes enriched. We have evaluated this method (see Figure 1) on β-hematin (a biocrystal with spectroscopic characteristics similar to hemozoin).10 We achieved an enhancement of nearly two orders of magnitude from the magnetic field enrichment. We have also determined that this methodology has a sensitivity of 5nM, which is equivalent to 30 parasites per μl in the ring stage (i.e., early stage of infection).

Figure 1. Surface-enhanced Raman spectroscopy (SERS) spectra of β-hematin that were obtained with (top) and without (bottom) the use of the magnetic-field enhancement methodology. Spectra are shown for β-hematin mixed with (a) and (d) iron oxide–silver ((Fe3O4@Ag) nanoparticles, without any nanoparticles (b) and (e), and (c) and (f) with (Fe3O4nanoparticles. PEx: Excitation power. a.u.: Arbitrary units.

More recently, we have developed another ultrasensitive approach that does not involve the use of an external magnet, and we have tested it on malaria-infected human blood.11 In this method, we synthesize Ag nanoparticles inside the malaria-infected erythrocytes (red blood cells) to realize a closer contact with hemozoin than in the traditional SERS technique, which involves simple mixing of the synthesized nanoparticles with isolated hemozoin. In our strategy we also lyse (i.e., cause breakdown of the cell membrane) the infected red blood cells so that we release their internal material without causing lysis of the actual parasites. We then dissolve silver nitrate in the parasite suspension so that Ag ions can enter the parasites. The Ag nanoparticles are subsequently reduced within the parasites.

We have evaluated the effectiveness of this new ultrasensitive strategy, in comparison to the traditional SERS approach, by testing it on human blood (see Figures 2 and 3). We find—see Figure 2—that the detection limit of the traditional SERS strategy is about 0.01% (i.e., about 500 parasites/μl). In contrast, the detection limit of our ultrasensitive method—see Figure 3—is much lower (about 0.00005%, or 2.5 parasites/μl). We note that this detection limit is even lower than that of the standard microscopy-based method (which has a detection limit of about 4–20 parasites/μl)3 and is close to that of PCR (limit of about 0.7 parasites/μl).5 With such a high sensitivity, our method could be used for the detection of single parasites.12

Figure 2. (a) SERS spectra of hemozoin in infected blood and (b) hemozoin contribution—as a function of parasitemia level—in the traditional SERS strategy. The spectra in (a) correspond to a range of different parasitemia levels and specific wavelength peaks are labeled with the Raman shift value. In (b), the data point corresponding to a parasitemia level of 0.01% (i.e., the detection limit of the methodology) is marked by a red box. The second-order polynomial line of best fit to the data points (between parasitemia levels of 0.01 and 0.2%) is also shown in red (equation of the line is also given). The normal blood (NB) data point has been added manually for comparison. A.U.: Arbitrary units.

Figure 3. (a) SERS spectra of normal and infected blood samples obtained with the use of the ultrasensitive strategy and (b) the hemozoin SERS peak intensity (at 1623cm-1) as a function of parasitemia level. The NB data point in (b) has been added manually for comparison.

In summary, we have developed two new SERS-based techniques to improve malaria detection methods. We have experimentally verified the effectiveness of both approaches and have shown their high sensitivity, which is suitable for early diagnosis of the malaria infection. In our current research we are working to facilitate the use of SERS techniques in the field. To that end, we are developing paper-based microfluidic chips that can be used to simplify sample preparation. In addition, we are developing a cost-effective spectrometer that can be used for the deployment of our new techniques in low-resource regions.

Keren Chen, Quan Liu
Nanyang Technological University
School of Chemical and Biomedical Engineering

Quan Liu received his PhD in biomedical engineering from the University of Wisconsin, Madison, and is currently an assistant professor. His research interests are focused on hyperspectral imaging and optical spectroscopy for medical diagnostics.

Aoli Xiong and Peter Preiser
School of Biological Science
Nanyang Technological University

1. http://www.who.int/malaria/publications/world-malaria-report-2015/report/en/ World Malaria Report 2015. Accessed 20 May 2016.
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6. B. R. Wood, S. J. Langford, B. M. Cooke, F. K. Glenister, J. Lim, D. McNaugton, Raman imaging of hemozoin within the food vacuole of Plasmodium falciparum trophozoites, Federat. Eur. Biochem. Soc. Lett. 554, p. 247-252, 2003.
7. N. L. Garrett, R. Sekine, M. W. A. Dixon, L. Tilley, K. R. Bambery, B. R. Wood, Bio-sensing with butterfly wings: naturally occurring nano-structures for SERS-based malaria parasite detection, Phys. Chem. Chem. Phys. 17, p. 21164-21168, 2015.
8. K. Chen, A. Xiong, C. Yuen, P. Preiser, Q. Liu, Towards field malaria diagnosis using surface enhanced Raman spectroscopy, Proc. SPIE 9887, p. 98870M, 2016. doi:10.1117/12.2227042
9. G. S. Noland, N. Briones, D. J. Sullivan Jr., The shape and size of hemozoin crystals distinguishes diverse Plasmodium species, Mol. Biochem. Parasitol. 130, p. 91-99, 2003.
10. C. Yuen, Q. Liu, Magnetic field enriched surface enhanced resonance Raman spectroscopy for early malaria diagnosis, J. Biomed. Opt. 17, p. 0170051, 2012. doi:10.1117/1.JBO.17.1.017005
11. K. Chen, C. Yuen, Y. Aniweh, P. Preiser, Q. Liu, Towards ultrasensitive malaria diagnosis using surface enhanced Raman spectroscopy, Sci. Rep. 6, p. 20177, 2016. doi:10.1038/srep20177
12. K. Chen, C. Perlaki, A. Xiong, P. Preiser, Q. Liu, Review of surface enhanced Raman spectroscopy for malaria diagnosis and a new approach for the detection of single parasites in the ring stage, IEEE J. Sel. Topics Quant. Electron. 22, p. 6900509, 2016. doi:10.1109/JSTQE.2016.2518959