On-Chip Magnetophoretic Concentration of Malaria-Infected Red Blood Cells and Hemozoin Nanocrystals

According to World Health Organization(WHO), 3.2 billion people are at risk for malaria. In 2015, 212 million new cases and 429000 deaths were estimated [1, 2]. Despite treatment in the early stage of the disease is usually very effective, conventional diagnostic tests via optical microscopy examination of thick and thin blood smears are unsuitable for an effective screening of the population. On the other hand, the over-treatment of the disease due to the large percentage of false positives in currently available rapid diagnostic tests (RDTs) may increase the risk of drug resistance. In this scenario, there is a strong need of novel RTDs with (i) the same sensitivity of the gold standard (optical microscopy examination) and (ii) a reduced number of false positives. To fulfill the last requirement, a real improvement would be to move from the detection of antigens or antibodies, which can be hardly washed out in a patient living in an endemic zone even after many weeks from the last malaria episode, to the quantification of infected red blood cells in a blood smear (i-RBC). This essentially means to go back to the concept of gold standard tests, with the additional requirement of integrating i-RBC counting in lab-on-chip platforms suitable for low-cost, rapid and on-site wide screening of the population in endemic zones. It is well known that i-RBCs display a paramagnetic behavior with respect to blood plasma, so that they can be separated from healthy ones and other corpuscles in a high magnetic field gradient. [3] This is due to the fact that, during the intra-erythrocytic development, the parasite degrades hemoglobin into free heme. This molecule, highly toxic to the parasite, is converted in an insoluble form, known as hemozoin or malaria pigment, which crystallizes into paramagnetic nanocrystals found both within the i-RBCs and free in the blood, after RBCs lysis. Of course, both the concentration of free hemozoin crystals and i-RBCs can be used for the determination of the parasitemia. In this paper we present an on-chip magnetophoretic platform for the separation and concentration of i-RBC and hemozoin nanocrystals on pre-defined areas of a chip. This is a pre-requisite for the quantification of the relative percentage of i-RBC with respect to healthy ones (parasitemia) with high sensitivity. The blood drop is placed on a glass substrate, which is then put in close contact to the surface of a chip with Nickel micropillars, at a distance defined by an outer ring which defines also the volume of the cell where magnetophoretic separation takes place. The chip is placed face-down, so that magnetic attraction towards the nickel pillars, in the macroscopic field gradient produced by an external system of permanent magnets, opposes the gravity. In this configuration, i-RBCs and hemozoin crystals are attracted upwards, towards the micropillars, while non-infected erythrocytes and the other blood cells (i.e. white blood cells and platelets) sediment towards the glass substrate. Our design allows to obtain a macroscopic field gradient as high as 1 × 10
¹⁵
A
²
/m
³
up to a distance of 500 micron from the chip surface, strong enough to overcome gravity and attract i-RBCs and hemozoin crystals towards the chip surface. The chip consists of an array of Ni pillars, with 20-30 micron diameter and 20 micron height, fabricated by electroplating and arranged on a hexagonal closed packed lattice. In close proximity to the chip surface, Ni pillars produce a much stronger field gradient, up to 3 × 10
¹⁶
A
²
/m
³
at a few microns from their surface, which concentrate the i-RBCs and hemozoin crystals. We have tested this system using RBCs from bovine blood, treated with NaNO
₂
in order to induce the transformation of hemoglobin into paramagnetic meta-hemoglobin. [4] In this way, we obtained suspensions in PBS of RBCs mimicking i-RBCs, suitable for experiments of capture. In figure 1 we report optical images from experiments performed in a direct configuration, where gravity and magnetic forces act in the same direction. Untreated RBCs (ut-RBCs) and treated ones with NaNO
₂
(t-RBCs) were stained with a red fluorophore to improve the quality of optical images. As evident from Figure 1, in case of t-RBCs we observed a capture efficiency of 100% by magnetic pillars in a lattice with center to center spacing of 80 microns. For hemozoin crystals the capture is even easier, because of their much larger volume susceptibility, 3.4 × 10
^-4
to be compared with 3.9 × 10
^-6
in case of i-RBCs. These results pave the way to the use of our magnetic chips as active slides for the magnetophoretic separation and concentration of malaria markers, both i-RBCs and hemozoin crystals, in well-defined areas of the chips where quantification can be performed with high sensitivity.