Dark field microscope-based single nanoparticle identification coupled with statistical analysis for ultrasensitive biotoxin detection in complex sample matrix

A novel approach for ultrasensitive ochratoxin A (OTA) detection is reported based on dark field microscope-based single nanoparticle identification coupled with a statistical analysis method. OTA aptamers were firstly hybridized with a single-stranded DNA (DNA1) to form an identification probe (DNA1-Apt). The aptamers separate from DNA1 in the presence of OTA and are released from the identification probe. Then, another single-stranded DNA (DNA2) hybridizes with DNA1 and result in the aggregation of gold nanoparticles (AuNPs). Therefore, the presence of AuNP aggregates is the evidence of the presence of OTA, while AuNP aggregates can be easily identified together with the monomers under dark field microscopic inspection. On the other hand, by counting the aggregation rate (the number of AuNP aggregates versus the number of AuNP monomers) with a statistical analysis method, OTA could be quantitatively detected. The detection range for OTA was 0.1 pg/mL ~ 30 ng/mL and the limit of detection was 0.1 pg/mL. The proposed sensor has comparative detection performance to sensors utilizing a number of signal amplification procedures, with the additional advantages of simplicity and high efficiency. The sensor can also be adopted for other target detection simply by replacing the identification probes. Graphical abstract The schematic of the AuNP aggregation for OTA detection. The OTA aptamers were competitively banded by OTA and induced form AuNP aggregation after adding DNA2 and AuNPs2. Subsequently, AuNPs were detected under dark field microscope and statistical analysis.