An ultrasensitive signal-on electrochemical aptasensor for ochratoxin A determination based on DNA controlled layer-by-layer assembly of dual gold nanoparticle conjugates

In this paper, a novel ultrasensitive signal-on electrochemical aptasensor has been proposed for Ochratoxin A (OTA) assay based on DNA controlled layer-by-layer assembly of dual gold nanoparticle (AuNP) conjugates. To construct the aptasensor, the 1 st AuNP conjugate was prepared by simultaneous immobilization of the capture probe 2 (CP2) and bridge probe (BP) onto the AuNPs. Then, OTA aptamer was loaded onto 1 st AuNPs by hybridization with CP2. The 1 st AuNP conjugate can be further immobilized onto the electrode by hybridization between BP and capture probe 1 (CP1), which was pre-immobilized on Au electrode. The 2 nd AuNP conjugate was prepared by immobilization of ferrocene (Fc) tagged SH-signal probe (SSP). Due to the recognition between aptamer on 1 st AuNP conjugate and OTA, CP2 was reformed in the ssDNA state, which can be utilized as the anchor for immobilization of 2 nd AuNP conjugate for electrochemical signal reporting. Because of the high surface-to-volume ratio and good conductivity of AuNPs, this dual AuNPs assembled nanoarchitecture finally lead to greatly improved abilities to load a large number of Fc molecules and significantly amply the electrochemical response even at a low target concentration. Both qualitative and quantitative analysis of OTA were thus realized by differential pulse voltammetry (DPV) signals, resulting in an excellent detection limit of 0.001 ppb and a wide dynamic range from 0.001 to 500 ppb over 6 orders of magnitude. Moreover, the real sample analysis towards OTA spiked wine samples was favorable, implying a great potential for practical applications. We envision that this unique dual AuNP conjugate assembly strategy would pave a new avenue for the development of versatile signal amplified electrochemical aptasensors.