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 1st 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 1st AuNPs by hybridization with CP2. The 1st 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 2nd AuNP conjugate was prepared by immobilization of ferrocene (Fc) tagged SH-signal probe (SSP). Due to the recognition between aptamer on 1st AuNP conjugate and OTA, CP2 was reformed in the ssDNA state, which can be utilized as the anchor for immobilization of 2nd 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.