Studies On Construction Of Ultrasensitive Gold Nanoparticles-based Aptasensor And Its Applications In Mycotoxin Detection

Mycotoxins are virulent secondary metabolites of fungi. They are the common contaminants in multifarious foods and feeds, threatening the safety of human beings and animals. High performance liquid chromatography(HPLC) and gas chromatography/mass spectrometry(GC-MS) are two prevalent approaches to determinate mycotoxins. Although these methods perform outstanding in sensitivity and limit of detection, they are not suitable for mycotoxins detection in our daily life, because of the requirement of expensive equipment, complicated operation procedures, and the well-trained professionals. Researchers from all over the world have devoted much efforts to developing a fast, inexpensive, highly sensitive and specific method. Particularly, nanoparticles play very important roles in signal generation and transduction. However, the reported methods are fail to realize both a broad assay range and low limit of detection(LOD) at the same time.To address above mentioned chalenges, this project develops two kinds of gold nanoparticles(Au NPs)-based aptasensor. Taken advantage of strengths of nano materials and oligonucleotides aptamer, novel aptasensors for mycotoxins detection with simple operation, high sensitivity and low cost have been successfully achieved.Research works and results are as follows.1. Development of gold nanoparticle(Au NP)-based AFB1 aptasensorAu NP owns a characteristic absorption peak at around 520 nm. In this work, AFB1 aptamer-conjugated Au NP acts as a recognition probe for AFB1 detection. Testing solution is first mixed with BSA-AFB1 in a certain concentration, into which the probe is added to start the analysis. In the absence of AFB1, aptamer will specifically bind to BSA-AFB1, resulting in aggregation or coagulation of Au NPs as well as the decrease of its absorbance at 520 nm. On the contrary, free AFB1 in the samples protects Au NPaptamer from binding to BSA-AFB1, further preventing the aggregation of Au NPs. Thus the change of absorbance at 520 nm is correlated with AFB1 concentration in a given sample. A limit of detection(LOD) of 4.1 pg m L-1 and a wide dynamic range from 10 pg m L-1 to 1 μg m L-1 are achieved for AFB1 detection with the developed aptasensor.2. Design and construction of FRET-based OTA aptasensorThe FRET-based aptasensor is composed of a free OTA aptamer, a Au NP coated with biotinylated partially complementary DNA(biotin-c DNA-Au NP), and a Cy3-conjugated streptavidin, each of which acts as a recognition molecule, energy acceptor, and energy donor, respectively. In the presence of OTA, aptamer detaches from its partially complementary DNA and binds to OTA, resulting in exposure of the biotin molecule located at the end of the partially complementary DNA., The Cy3-conjugated streptavidin is attached to Au NP surface based on the specific interaction between biotin and streptavidin. Thus FRET from Cy3 to Au NP subsequently happens due to the very close distance. With the unique design, an LOD of 1.4 pg m L-1 and a wide assay range from 2.5 pg m L-1 to 1 μg m L-1 are achieved for OTA detection.3. Specificity and selectivity analysis of the developed aptasensorsTwo common mycotoxins FB1 and DON were utilized as interferences. In addition, the inter-interference between AFB1 and OTA was also evaluated. Only significant responses were observed from the corresponding targets of these two aptasensors, while negligible signal was obtained from other mycotoxins. These results demonstrate that the developed aptasensors have good selectivity and specificity.4. Evaluation of their potential application in analysis of real samplesExtractives from wheat and green coffee beans were prepared as mimic samples to evaluate the potential of the aptasensor for practical application. OTA standard solutions with different concentrations are spiked into the above extractive and analyzed with the developed aptasensor. Although LOD from the mimic samples is poorer than that observed from lab buffer, it is still less than tenth of the maximum limit permitted by China and European Commission. The experimental results demonstrate a great feasibility for analysis of various real samples with the developed aptasensors.In summary, two Au NP-based aptasensors with high-performance were successfully developed for mycotoxins detection. These aptasensors with special designs take good advantage of the superiorities of Au NP and aptamer. Highperformance with simple operation, high sensitivity and good specificity are achieved for detection of AFB1 and OTA in both lab buffer. Besides, OTA aptase9 nsors achieve ideal result in extractive from wheat and green coffee bean, demonstrating its great potential in practical applications. This project not only provides an sensitive and effective aptasensors for mycotoxins detection, but also opens up a new way for detection of small molecules in various fields.