Refined Method for Droplet Microfluidics-Enabled Detection of Plasmodium falciparum Encoded Topoisomerase I in Blood from Malaria Patients
Marianne Smedegaard Hede,
Patricia Nkem Okorie,
Signe Kirk Fruekilde,
Søren Fjelstrup,
Jonas Thomsen,
Oskar Franch,
Cinzia Tesauro,
Magnus Tobias Bugge,
Mette Christiansen,
Stéphane Picot,
Felix Lötsch,
Ghyslain Mombo-Ngoma,
Johannes Mischlinger,
Ayôla A. Adegnika,
Finn Skou Pedersen,
Yi-Ping Ho,
Eskild Petersen,
Magnus Stougaard,
Michael Ramharter,
Birgitta Ruth Knudsen
Affiliations
Marianne Smedegaard Hede
Zymonostics, 8000 Aarhus C, Denmark
Patricia Nkem Okorie
Institute for Advanced Medical Research and Training, College of Medicine, University of Ibadan, 200001 Ibadan, Nigeria
Signe Kirk Fruekilde
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Søren Fjelstrup
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Jonas Thomsen
Zymonostics, 8000 Aarhus C, Denmark
Oskar Franch
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Cinzia Tesauro
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Magnus Tobias Bugge
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Mette Christiansen
Department of Clinical Immunology, Aarhus University Hospital, 8200 Aarhus N, Denmark
Stéphane Picot
Malaria Research Unit, SMITh, ICBMS, UMR 5246 CNRS-INSA-CPE-UCBL1 & Institutde Parasitologie et Mycologie Médicale, Hospices Civils de Lyon, 69373 Lyon Cedex 08, France
Felix Lötsch
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon
Ghyslain Mombo-Ngoma
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon
Johannes Mischlinger
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon
Ayôla A. Adegnika
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon
Finn Skou Pedersen
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Yi-Ping Ho
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Eskild Petersen
Department of Infectious Diseases, Aarhus University Hospital, 8200 Aarhus N, Denmark
Magnus Stougaard
Department of Clinical Medicine, University of Aarhus, 8000 Aarhus C, Denmark
Michael Ramharter
Centre de Recherches Médicales de Lambaréné, Lambaréné, Gabon
Birgitta Ruth Knudsen
Department of Molecular Biology and Genetics, University of Aarhus, 8000 Aarhus C, Denmark
Rapid and reliable diagnosis is essential in the fight against malaria, which remains one of the most deadly infectious diseases in the world. In the present study we take advantage of a droplet microfluidics platform combined with a novel and user-friendly biosensor for revealing the main malaria-causing agent, the Plasmodium falciparum (P. falciparum) parasite. Detection of the parasite is achieved through detection of the activity of a parasite-produced DNA-modifying enzyme, topoisomerase I (pfTopoI), in the blood from malaria patients. The assay presented has three steps: (1) droplet microfluidics-enabled extraction of active pfTopoI from a patient blood sample; (2) pfTopoI-mediated modification of a specialized DNA biosensor; (3) readout. The setup is quantitative and specific for the detection of Plasmodium topoisomerase I. The procedure is a considerable improvement of the previously published Rolling Circle Enhanced Enzyme Activity Detection (REEAD) due to the advantages of involving no signal amplification steps combined with a user-friendly readout. In combination these alterations represent an important step towards exploiting enzyme activity detection in point-of-care diagnostics of malaria.
