Dataset Information

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

ABSTRACT: We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing Plasmodium species in single drops of unprocessed blood with a detection limit of less than one parasite/?L. Moreover, the setup allowed for detection of Plasmodium parasites in noninvasive saliva samples from infected patients. During recent years malaria transmission has declined worldwide, and with this the number of patients with low-parasite density has increased. Consequently, the need for accurate detection of even a few parasites is becoming increasingly important for the continued combat against the disease. We believe that the presented droplet microfluidics platform, which has a high potential for adaptation to point-of-care setups suitable for low-resource settings, may contribute significantly to meet this demand. Moreover, potential future adaptation of the presented setup for the detection of other microorganisms may form the basis for the development of a more generic platform for diagnosis, fresh water or food quality control, or other purposes within applied or basic science.

SUBMITTER: Juul S

PROVIDER: S-EPMC3528816 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

Juul Sissel S Nielsen Christine J F CJ Labouriau Rodrigo R Roy Amit A Tesauro Cinzia C Jensen Pia W PW Harmsen Charlotte C Kristoffersen Emil L EL Chiu Ya-Ling YL Frøhlich Rikke R Fiorani Paola P Cox-Singh Janet J Tordrup David D Koch Jørn J Bienvenu Anne-Lise AL Desideri Alessandro A Picot Stephane S Petersen Eskild E Leong Kam W KW Ho Yi-Ping YP Stougaard Magnus M Knudsen Birgitta R BR

ACS nano 20121107 12

We present an attractive new system for the specific and sensitive detection of the malaria-causing Plasmodium parasites. The system relies on isothermal conversion of single DNA cleavage-ligation events catalyzed specifically by the Plasmodium enzyme topoisomerase I to micrometer-sized products detectable at the single-molecule level. Combined with a droplet microfluidics lab-on-a-chip platform, this design allowed for sensitive, specific, and quantitative detection of all human-malaria-causing ...[more]

PMID: 23121492

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Project description:BackgroundCustom synthesized DNA is in high demand for synthetic biology applications. However, current technologies to produce these sequences using assembly from DNA oligonucleotides are costly and labor-intensive. The automation and reduced sample volumes afforded by microfluidic technologies could significantly decrease materials and labor costs associated with DNA synthesis. The purpose of this study was to develop a gene assembly protocol utilizing a digital microfluidic device. Toward this goal, we adapted bench-scale oligonucleotide assembly methods followed by enzymatic error correction to the Mondrian™ digital microfluidic platform.ResultsWe optimized Gibson assembly, polymerase chain reaction (PCR), and enzymatic error correction reactions in a single protocol to assemble 12 oligonucleotides into a 339-bp double- stranded DNA sequence encoding part of the human influenza virus hemagglutinin (HA) gene. The reactions were scaled down to 0.6-1.2 ?L. Initial microfluidic assembly methods were successful and had an error frequency of approximately 4 errors/kb with errors originating from the original oligonucleotide synthesis. Relative to conventional benchtop procedures, PCR optimization required additional amounts of MgCl2, Phusion polymerase, and PEG 8000 to achieve amplification of the assembly and error correction products. After one round of error correction, error frequency was reduced to an average of 1.8 errors kb-?1.ConclusionWe demonstrated that DNA assembly from oligonucleotides and error correction could be completely automated on a digital microfluidic (DMF) platform. The results demonstrate that enzymatic reactions in droplets show a strong dependence on surface interactions, and successful on-chip implementation required supplementation with surfactants, molecular crowding agents, and an excess of enzyme. Enzymatic error correction of assembled fragments improved sequence fidelity by 2-fold, which was a significant improvement but somewhat lower than expected compared to bench-top assays, suggesting an additional capacity for optimization.

Dataset Information

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

Publications

Droplet microfluidics platform for highly sensitive and quantitative detection of malaria-causing Plasmodium parasites based on enzyme activity measurement.

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