The development of rapid, accurate, and efficient detection methods for protozoan parasites can substantially control the outbreak of protozoan parasites infection, which poses a threat to global public health. Idealistically, electrochemical biosensors would be able to overcome the limitations of current detection methods due to their simplified detection procedure, on-site quantitative analysis, rapid detection time, high sensitivity, and portability. The objective of this scoping review is to evaluate the current state of electrochemical biosensors for detecting protozoan parasites. This review followed the most recent Preferred Reporting Items for Systematic Review and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) recommendations. Using electrochemical biosensor and protozoan parasite keywords, a literature search was conducted in PubMed, Scopus, Web of Science, and ScienceDirect on journals published between January 2014 and January 2022. Of the 52 studies, 19 were evaluated for eligibility, and 11 met the review's inclusion criteria to evaluate the effectiveness and limitations of the developed electrochemical biosensor platforms for detecting protozoan parasite including information about the samples, biomarkers, bioreceptors, detection system platform, nanomaterials used in fabrication, and limit of detection (LoD). Most electrochemical biosensors were fabricated using conventional electrodes rather than screen-printed electrodes (SPE). The range of the linear calibration curves for the developed electrochemical biosensors was between 200 ng/ml and 0.77 pM. The encouraging detection performance of the electrochemical biosensors demonstrate their potential as a superior alternative to existing detection techniques. On the other hand, more study is needed to determine the sensitivity and specificity of the electrochemical sensing platform for protozoan parasite detection.
Amoebiasis, caused by Entamoeba histolytica, remains a global health concern due to its asymptomatic nature and potential for severe complications such as liver abscesses. To investigate molecular recognition mechanisms, this study focused on the binding affinity and detection limit of aptamers targeting E. histolytica actin (EhActin), a cytoskeletal protein selected as a target molecule. A genomic tRNA-based sequence library was computationally screened to identify potential high-affinity aptamer candidates, followed by molecular docking and molecular dynamics simulations to evaluate interaction profiles. Recombinant EhActin (rEhActin) was expressed in Escherichia coli LEMO21 using a pET28a(+) vector system and used in ELONA to quantify the interaction between a biotinylated DNA aptamer and rEhActin. The limit of detection was also assessed using ELONA with E. histolytica protein extract. Among the shortlisted aptamers, APT29 demonstrated favorable in silico and in vitro binding characteristics. Molecular dynamics analysis supported overall structural stability of the APT29-EhActin complex, although hydrogen bond interactions fluctuated over time. ELONA showed a dissociation constant (Kd) of 44.72 nM for APT29, and this aptamer achieved a limit of detection of 0.4 µg/well of E. histolytica protein extract. These findings suggest APT29 as a promising molecular probe for further biophysical and structural studies of aptamer-target interactions involving E. histolytica, with potential relevance to future diagnostic development for amoebiasis.