Development of a polyaniline/reduced graphene oxide composite electrode for harmful algal bloom DNA biosensing

Abstract

Current methods for monitoring Harmful Algal Bloom (HAB) dinoflagellates, including PCR and microscopic techniques, are inefficient and slow. These methods are often species-specific and confined to laboratories, hampering timely preventive action. Hence, this study focuses on four objectives: analyse HAB occurrences over the past two decades, select suitable DNA sequences and nanomaterials for biosensing, develop a sensitive nanomaterial-based DNA electrode, and validate the electrochemical DNA biosensor against traditional cell counting microscopy for accurate HAB analysis of toxigenic algae in seawater samples. The methodology involved examining HAB cases in Malaysia, assessing their impact, location, and management. In silico studies were performed to select a DNA probe effective for generic HAB species. The nanohybrid

polyaniline/graphene (PGN) composite was properly synthesized through reverse- phase polymerization. The DNA biosensor's accuracy was validated using microscopic

cell counting, and quantitative data were analysed through a recovery assessment approach. Results show that food poisoning cases due to HABs were observed in 2014 and earlier, with subsequent yearly reports highlighting losses from fish deaths and seafood contamination. In Malaysia, microscopic techniques are the primary method for monitoring HAB events. Using BioEdit and Blast software, a 38-mers DNA sequence from the sxtA4 gene was selected with 100% identity to saxitoxin HAB species. The well-dispersed PGN with an identified ideal loading of 0.5 mg mL-1 enhanced the sensitivity of the electrochemical voltammetric signal. The optimised DNA biosensor, with a 5-hour probe immobilization duration, 0.2 M ionic strength, pH 7.5 incubation media, and a DNA hybridization temperature of 60 °C, accurately detected A. minutum in a linear concentration of 10 to 10,000,000 cells L-1

. Validation against microscopic counting methods showed a high recovery percentage (80–120%). Additionally, the biosensor successfully quantified another saxitoxin-producing species, A. tamiyavanichii, while showing distinct signals to a harmless microalgae species, Isochrysis galbana. In conclusion, the developed electrochemical DNA biosensor holds promise as a single tool for monitoring harmful saxitoxin-producing HAB species, functioning as an early warning system with a quick 30-minute response time. The research lays the foundation for a portable on-site detection device, potentially revolutionizing HAB monitoring and advancing biosensor technology.