Development of Aptamer-Based Point-of-Care Diagnostic Devices for Malaria Using Three-Dimensional Printing Rapid Prototyping

Abstract

We present the adaption of an aptamer-tethered enzyme capture (APTEC) assay into point-of-care device prototypes with potential for malaria diagnosis. The assay functions by capturing the malaria biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) from samples and using its intrinsic enzymatic activity to generate a visualizable blue color in response to Plasmodium positive samples. Using three-dimensional (3D) printing rapid prototyping, a paper-based syringe test and magnetic bead-based well test were developed. Both were found to successfully detect recombinant PfLDH at ng mL–1 concentrations using low sample volumes (20 μL) and could function using purified or spiked whole blood samples with facile sample preparation. The syringe test was found to be more analytically sensitive but required more additional preparation steps, while the well test required fewer steps and hence may be better suited for future clinical testing. Additionally, the development reagents required for the color response were fully stabilized through desiccation with sugar stabilization agents and could withstand temperatures as high as 90 °C. This study demonstrates how a previously reported biochemical assay can be adapted into workable point-of-care device prototypes by using 3D printing rapid prototyping. This novel technology, intended for rapid diagnostic tests (RDTs) for malaria, is distinct and carries many potential advantages relative to established lateral flow immunochromatographic approaches.

Recent Advances in the Development of Biosensors for Malaria Diagnosis

“…Aptamer functionalized microbeads were used to determine the capture and measure the intrinsic enzymatic activity of LDH in a colorimetric assay ( Figure 4A) [63]. In the aptamer-tethered enzyme capture (APTEC) assay, the beads conferred a wide surface area for analyte binding to produce a LoD of 4.9 ng/mL for recombinant PfLDH (Table 1).…”

“…Application of colloidal AuNPs functionalized with HRPlabelled detection antibodies enhanced amperometric signals to LoDs down to 19 pg/mL (in buffer) and 23 pg/mL (in serum) ( Table 1). Aptamer functionalized microbeads were used to determine the capture and measure the intrinsic enzymatic activity of LDH in a colorimetric assay ( Figure 4A) [63]. In the aptamer-tethered enzyme capture (APTEC) assay, the beads conferred a wide surface area for analyte binding to produce a LoD of 4.9 ng/mL for recombinant PfLDH (Table 2).…”

Simple, rapid, and accurate malaria diagnostic platform using microfluidic-based immunoassay of Plasmodium falciparum lactate dehydrogenase

“…Table 1 summarizes and compares recent studies on the diagnosis of PfLDH. Recently, many studies have developed high-sensitivity, high-selectivity, and cost-effective diagnostics based on aptamers or nanoparticles [31][32][33][34][35]. Nanoparticles, such as novel-metal nanoparticles and magnetic nanoparticles, are applied for lateral flow immunoassay or combined with aptamers to improved diagnostic performance.…”

“…After the reaction, BSA 1% in PBS buffer (100 μL) was added to the wells and reacted at 37 °C for 30 min. After the removal of BSA solution, PfLDH (100 μL dissolved in PBS) was added to the wells in a concentration range of 1 μg/mL to [35] 100 μg/mL and reacted at 37 °C for 120 min. After PfLDH treatment, rabbit polyclonal PfLDH antibody as a 1st-Ab (100 μL, 1/2000 dilution) was immediately added onto the PfLDH solution.…”

Aptamer‐Mediated Protein Molecular Recognition Driving a DNA Tweezer Nanomachine

“…We have previously developed a DNA aptamer against the malaria diagnostic biomarker Plasmodium falciparum lactate dehydrogenase (PfLDH) . We have shown how the aptamer can be used in complex samples, integrated within a 3D printed module to make a portable point‐of‐care device . Previously, we have also shown how the aptamer remains functional within a DNA origami assembly .…”