A multidisciplinary approach to develop aptamer-based biosensors for malaria diagnosis

Abstract

The challenge of malaria is multifaceted and complex, yet over the last decade the rate of mortality has halved. Much of the progress is attributed to the effective utilisation of preventative methods e.g. pesticide treated bed nets, improved methods of diagnosis, the increased availability of primary care facilities, and the effective use drug treatments. Despite all of these advances, malaria still kills more than 400,000 people a year. This study focuses on the utilisation of novel technologies to improve the diagnosis of malaria.

Current portable diagnostic tests are based on thermally unstable antibody technologies. Many antibody-based diagnostic tests are ineffective if they are not transported and stored in a thermally controlled supply chain. Therefore, there is an urgent need to integrate thermally stable technologies into portable diagnostic tests. The aptamer tethered enzyme capture (APTEC) assay is a laboratory-based assay which uses aptamers as an alternative to antibodies. Aptamers are thermally stable and more conducive to the supply transport in malaria endemic regions.

This study describes how the APTEC assay was integrated into a 3D printed portable microfluidic biosensor for the point of care diagnosis of malaria. A strategy of rapid prototyping by 3D printer successfully integrated and optimised each step of the lab-based APTEC assay onto a portable magnet controlled microfluidic cassette with the dimensions 40mm X 25mm X 2mm. The smallest dimension on cassette was 200μm and the height of each chamber and channel was sub-millimetre (500μm). Two image analysis techniques were used to quantify the colorimetric signal on the cassette, the most successful analysis method, the ‘Pixel Area Method’ detected synchronous 3D7 P. falciparum malaria parasites in human blood at concentrations as low as 250 parasite/μL, surpassing the sensitivity of the lab - based APTEC assay. Furthermore, the portable microfluidic biosensor was specific for 100% of P. falciparum containing patient samples versus uninfected controls and detected 90% of P. falciparum infected patient samples. The aptamer in the biosensor is specific for P. falciparum but the biosensor non-specifically detected parasites in two of patient samples infected with of P. vivax.

The project was then expanded and a portable high-throughput detection system for the APTEC assay was developed. First, a portable spectrophotometer was designed to measure assay colorimetric signal. Second, a portable and modular magnetic transfer tool was prototyped by 3D printing for high-throughput magnetic bead-based assays. Both tools used together detected recombinant P. falciparum Lactate Dehydrogenase in simulated blood samples with a Limit of Detection of 37-57 ng/mL.

Next, to increase biomarker capture capacity in the APTEC assay, aptamer supramolecular nanostructure hybrids were functionalised on magnetic beads. Although the nanostructure coated beads were functional in the APTEC assay they did not improve assay capture capacity relative to the standard aptamer coated beads. Thereafter, Aptamer DNA tetrahedron nanostructures were utilised, which provided a 6-fold improvement to the APTEC assay.

In conclusion, a multidisciplinary approach improved the portability, throughput, and sensitivity of a thermally stable laboratory-based assay for the diagnosis of malaria.