Aptamer-Based Electrochemical Microfluidic Biosensor for the Detection of Cryptosporidium parvum

Cryptosporidium parvum is a high-riskand opportunistic waterborne parasitic pathogen with highly infectiousoocysts that can survive harsh environmental conditions for long periods.Current state-of-the-art methods are limited to lengthy imaging andantibody-based detection techniques that are slow, labor-intensive,and demand trained personnel. Therefore, the development of new sensingplatforms for rapid and accurate identification at the point-of-care(POC) is essential to improve public health. Herein, we propose anovel electrochemical microfluidic aptasensor based on hierarchical3D gold nano-/microislands (NMIs), functionalized with aptamers specificto C. parvum. We used aptamers as robustsynthetic biorecognition elements with a remarkable ability to bindand discriminate among molecules to develop a highly selective biosensor.Also, the 3D gold NMIs feature a large active surface area that provideshigh sensitivity and a low limit of detection (LOD), especially whenthey are combined with aptamers,. The performance of the NMI aptasensorwas assessed by testing the biosensor's ability to detect differentconcentrations of C. parvum oocystsspiked in different sample matrices, i.e., buffer, tap water, andstool, within 40 min detection time. The electrochemical measurementsshowed an acceptable LOD of 5 oocysts mL(-1) in buffermedium, as well as 10 oocysts mL(-1) in stool andtap water media, over a wide linear range of 10-100,000 oocystsmL(-1). Moreover, the NMI aptasensor recognized C. parvum oocysts with high selectivity while exhibitingno significant cross-reactivity to other related coccidian parasites.The specific feasibility of the aptasensor was further demonstratedby the detection of the target C. parvum in patient stool samples. Our assay showed coherent results withmicroscopy and real-time quantitative polymerase chain reaction, achievinghigh sensitivity and specificity with a significant signal difference(p < 0.001). Therefore, the proposed microfluidicelectrochemical biosensor platform could be a stepping stone for thedevelopment of rapid and accurate detection of parasites at the POC.