Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer.

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/µL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

Quantitative Detection of Plasmodium falciparum Using, LUNA-FL, A Fluorescent Cell Counter.

The microscopic examination of Giemsa-stained thin and/or thick blood films (Giemsa microscopy) is the standard method of malaria diagnosis. However, the results of the diagnosis significantly depend on the skills of clinical technicians. Furthermore, sample preparation and analysis are laborious and time-consuming. Therefore, in this study, we investigated if a commercially available fluorescent cell counter, LUNA-FL, was useful for the detection of Plasmodium parasite and the estimation of parasitemia. Whole blood samples from uninfected persons, spiked with P. falciparum-infected erythrocytes, were analysed. Most of the leucocytes and platelets were removed from whole blood samples with SiO2-nanofiber filters set on spin columns. The filtered samples were stained with acridine orange, and automatic detection, as well as counting of erythrocytes and parasites, were performed using LUNA-FL. Whole blood, with various levels of parasites, was analysed by Giemsa microscopy or with LUNA-FL to estimate parasitemia, and a comparative analysis was performed. The coefficient determination value of the regression line was high (R2 = 0.98), indicating that accurate quantitative parasite detection could be performed using LUNA-FL. LUNA-FL has a low running cost; it is compact, fast, and easy to operate, and may therefore be useful for point-of-care testing in the endemic areas.

Application of a Glucose Dehydrogenase-Fused with Zinc Finger Protein to Label DNA Aptamers for the Electrochemical Detection of VEGF.

Aptamer-based electrochemical sensors have gained attention in the context of developing a diagnostic biomarker detection method because of their rapid response, miniaturization ability, stability, and design flexibility. In such detection systems, enzymes are often used as labels to amplify the electrochemical signal. We have focused on glucose dehydrogenase (GDH) as a labeling enzyme for electrochemical detection owing to its high enzymatic activity, availability, and well-established electrochemical principle and platform. However, it is difficult and laborious to obtain one to one labeling of a GDH-aptamer complex with conventional chemical conjugation methods. In this study, we used GDH that was genetically fused to a DNA binding protein, i.e., zinc finger protein (ZF). Fused GDH can be attached to an aptamer spontaneously and site specifically in a buffer by exploiting the sequence-specific binding ability of ZF. Using such a fusion protein, we labeled a vascular endothelial growth factor (VEGF)-binding aptamer with GDH and detected the target electrochemically. As a result, upon the addition of glucose, the GDH labeled on the aptamer generated an amperometric signal, and the current response increased dependent on the VEGF concentration. Eventually, the developed electrochemical sensor proved to detect VEGF levels as low as 105 pM, thereby successfully demonstrating the concept of using ZF-fused GDH to enzymatically label aptamers.

Enzyme linking to DNA aptamers via a zinc finger as a bridge.

We propose a novel enzyme-labeling method for DNA aptamers using enzyme-fused zinc finger proteins. We achieved thrombin detection and vascular endothelial growth factor detection using zinc finger-fused firefly luciferase.