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Title: Lab-on-chip for magnetic resonance relaxometry detection for rapid malaria diagnosis
Authors: Kong, Tian Fook
Keywords: DRNTU::Engineering
Issue Date: 2014
Abstract: Malaria is an infectious disease that affects 3.4 billion people, especially in the tropical and subtropical countries such as Africa and South-East Asia. The major challenges in current clinical malaria diagnostics are in obtaining a sensitive, robust, fast, and inexpensive measurement from patient blood sample. While one of the leading causes of the high mortality rate in malaria cases is the delay in medical diagnosis and treatment. Prompt diagnosis and timely treatment for malaria is often difficult to come by in malaria endemic area. Most malaria deaths can be prevented if diagnosis and effective treatment were administered within 24 hour after the onset of the first symptom. Therefore, access to fast, sensitive and reliable diagnostic tool is of inevitable importance. Rapid diagnostic tests (RDTs) such as the dipsticks and quantitative buffy coat were developed to aid the detection of malaria. However, negative RDTs results cannot be accepted directly without confirmation by microscopic examination. At present, Giemsa-stained microscopic examination of thick blood smear remains the gold standard for laboratory diagnosis of malaria. Although the procedure is labor intensive, time consuming and requires a well-trained and highly experienced personnel, Giemsa microscopic examination is still being practiced in the current clinical setting for more than a century. The aim of this work is to develop an inexpensive novel malaria diagnosis method using magnetic resonance relaxometry (MRR) technique for the rapid detection and quantification of malaria infected red blood cells. During the intra-erythrocytic cycle, malaria parasites metabolize large amount of cellular hemoglobin and convert them into hemozoin crystallites or malaria pigment that is paramagnetic. These paramagnetic malaria pigments serve as the natural bio-marker for malaria detection with MRR measurement. The bulk magnetic susceptibility of the infected red blood cells (iRBCs) is significantly higher than the healthy red blood cells (hRBCs) due to the presence of the paramagnetic crystallites. Therefore, the iRBCs have higher transverse relaxation rate, R2. The detection of the subtle increase in the R2 due to the presence of malaria parasites forms the basis of the MRR malaria detection. The performance and limit of detection of the bench-top MRR system were studied with malaria infected human (P. falciparum) and mouse blood (balb/c, P. berghei). The MRR malaria detection system, as demonstrated in the dilution tests, could potentially achieve a limit of detection of 0.0005% parasitemia level, depending on the stage of malaria infection. Nonetheless, MRR detection has a major drawback in that it measures the absolute value of a blood sample instead of detecting a relative change in the R2 value. In other words, MRR system alone could not function as a malaria diagnosis tool unless the baseline or the healthy value of the blood R2 of the particular individual is previously known or measured before malaria infection. In order to mitigate the R2 baseline restriction, the iRBCs are separated from the hRBCs through a deformability based separation method known as margination, and subsequently, the relative changes in the relaxation rates of the separated blood samples are compared. Margination is a naturally occurring hemodynamic phenomenon in our blood vessels where the stiffer nucleated white blood cells (leukocytes) are segregated towards the vessel wall to promote leukocytes adhesion and extravasation. The same margination principle could also be applied to marginate stiffer malaria iRBCs towards the side-walls. The margination efficiency of the mulitplex high throughput margination device that contains 7 parallel margination channels are evaluated. The iRBCs in all infection stages could be effectively separated from hRBCs with a parasitemia enrichment factor of 1.9 ± 0.2, 4.1 ± 0.8, and 32.1 ± 7.6 for ring, trophozoite, and schizont, respectively. In addition, the flow rate analysis reveals that increasing the margination flow rates from 1 to 10 µL min-1 does not adversely affect the margination performance significantly. Hence, the flow rate could be increased to improve the throughput of the margination system without compromising the separation performance. Furthermore, the microfluidic margination device not only separates the iRBCs from hBCS, the stiffer old hRBCs are also being separated from the more deformable young hRBCs. RBCs are naturally damaged by oxidation during aging and its stiffness increases substantially. As a result, the side outlet R2 value is always significantly higher than the inlet, while the middle R2 is lower than the inlet R2 for hRBCs. Nonetheless, lysing the RBCs prior to the MRR measurements diminishes the relative difference in R2 relaxation rates between the blood samples collected at the inlet and middle. Henceforth, the malaria detection will be based on comparing the relative R2 values of the lysed blood samples between the inlet and middle. As the iRBCs are marginated towards the sidewalls, the middle outlet is depleted of iRBCs. The middle R2 values would be significantly lower (with P-value < 0.05) compared to the inlet with the presence of iRBCs, and non-distinguishable statistically (with P-value > 0.05) if the sample contains only hRBCs. The novel diagnostic method proposed here offers rapid malaria diagnosis with a detection sensitivity that is comparable to PCR, which is one order of magnitude more sensitive than the standard Giemsa microscopic method. The transverse relaxation rate, R2 value of the lysed separated early ring stage iRBCs at extremely low parasitemia count of 0.0005% significantly differs from the hRBCs with P < 0.05. In the near future, a Lab-on-a-Chip solution with MRR functionality integrated is envisaged for malaria diagnosis in the clinical setting. As the first step of working towards the goal of realizing an integrated MRR chip, a novel technique for the fabrication of three-dimensional multilayer liquid-metal microcoils by lamination of dry adhesive sheets were developed. The integrated liquid-metal NMR device consists of two major components: a microcoil and a sample chamber. The fabrication of the integrated device is achieved by stacking or lamination of multiple layers of laser-cut adhesive sheets. The lamination of laser cut dry adhesive sheets offers a convenient way to fabricate three-dimensional microcoil without considerable fabrication efforts and the need of incurring high fabrication cost. The liquid-metal microcoil readily integrates with the sample chamber for realizing a lab-on-a-chip platform for MRR measurements. From the microcoil characterization experiments, the ability of liquid-metal microcoil for performing MRR measurements are demonstrated. The results obtained from the microcoil for the parametric study of the hematocrit- R2 relationship are in good agreement with the data obtained with the conventional solenoid-type coil. The integrated microcoil has a low direct-current resistance of 0.3 Ω and a relatively high inductance of 67.5 nH leading to a high quality factor of approximately 30 at 21.65 MHz.
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