Novel Ootical Nano Biosensors For Detection Of Mycotoxins In Agro-Products

The damage of crops and agro-products caused by mycotoxins induces serious economic losses directly and indirectly on a global scale. Many mycotoxins can cause serious harm to human health and even death in people through mycotoxin-contaminated food, especially aflatoxins and ochratoxins which are most toxic and polluted. Traditional detection methods such as thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and enzyme-linked immuno sorbent assay (ELISA) are sensitive; however, more rapid, simple, and cost-effective approaches are still requested by the food industry. More recently optical biosensors coupled with metal nanoparticles provides a promising potential approach for the purpose, which is perhaps one of the most powerful and simple nanosensing methods available. Wherein gold nanorods (GNRs) as one kind of anisotropy one-dimensional materials, have received much attention in the past decade due to their unique optical properties.In this study, aflatoxin B1(AFB1) and ochratoxin A (OTA) in common agro-products like peanuts and grain were selected as detection target of interest. Biosensing methods and quantitative detection models based on localized surface plasmon resonance (LSPR) absorption spectra and dynamic light scattering (DLS) of GNRs have been established, with biological recognition elements of specific monoclonal antibody and aptamer. It is the first time to explore the available biosensing approaches based on GNRs for rapid and simple discrimination of mycotoxins, providing a new platform for on-site testing of mycotoxin contamination in common agro-products.The main contents and results are summarized as follows:(1) The physical and chemical properties of GNRs were studied initially. Various techniques including transmission electron microscopy (TEM), UV-vis-NIR absorption spectra, dynamic light scattering and inductively coupled plasma mass spectrometry (ICP-MS) were used for size, hydrodynamic diameter, concentration and optical property characterization of GNRs. The results of stability study showed that:GNRs could keep stable in CTAB solution of different pH environment (pH value from2to13); higher stability of GNRs could been observed under strong ionic strength conditions at a higher CTAB concentration, and GNRs got irreversible aggregation while mixing with NaCl solution concentration greater than160mM; GNRs could keep stable in biological analyzing buffer of phosphate buffered saline (PBS) with concentration in the range of0-10mM or greater than100mM. It provided theoretical basis for analysis and application based on GNRs.(2) Different biosensing method based on end and satellite antibody modified GNRs were established and compared, using alpha-fetoprotein (AFP) as a model analyte which is one of the major markers for hepatocellular tumors. Direct immune sensing method with end modified GNRs was investigated. Change in refractive index of surrounding environment after AFP binding was recognized according to shift of longitudinal LSPR wavelength, however the linear detection range of this method was narrow (0.25-1.0nM) with high requirement of equipment in resolution and signal to noise ratio. Absorption intensity of normalized spectra could also been used for AFP sensing (0.25-14.3nM), however the detection performance was not ideal. For immune sensing approach with satellite modified GNRs, dramatic aggregation of GNRs was induced by the interaction between target proteins and capture antibodies. Associated wavelength red-shifts and intensity decreases in the absorption peak of GNRs were observed with increasing concentrations of target AFP. It was illustrated that this method can be used reliably to detect AFP protein in PBS solution at concentrations of picomolar level with a wide quantification range from0.25to4.0nM. The limit of detection and quantification based on wavelength shift was0.18and0.60nM, and based on absorption intensity was0.04and0.14nM, respectively. The complication of interfering proteins in the sample showed negligible effects on the detection results. It is expected that plamon coupling based signal enhancement caused by GNR aggregation would be applicable for discrimination of mycotoxins in agro-products at low concentrations.(3) A promising one-step and label-free optical biosensor was illustrated for determination of AFB1that is most commonly found in foods and highly dangerous even at very low concentrations. In this research, GNRs were employed as a sensing platform, which showed high stability under high ionic strength conditions without addition of any stabilizing agent. GNR-AFB1-BSA (bovine serum albumin) conjugates aggregated after mixing with free antibodies, resulting in significant changes in absorption intensity. At the same time the existence of AFB1molecules in samples caused dispersion of nanorods, as a result of competitive immune-reaction with antibodies. By taking advantages of the competitive dispersion of GNRs, the developed method could effectively reduce false results caused by undesirable aggregation, which is a big problem for spherical gold nanoparticles. Absorption intensity of UV-vis spectra served as the sensing indicator, with dynamic light scattering measurement as another sensing tool. While the concentration of AFB1antibody was15μg/mL, the biosensing system could detect AFB1standard PBS solution linearly from0.1to1.0ng/mL, with a low limit of detection (LOD) of0.035ng/mL. While the concentration of AFB1antibody was25μg/mL, the designed biosensing system could detect AFB1in a linear range from0.5to20ng/mL, with a good correlation coefficient of0.99. And the limit of detection was0.16ng/mL, indicating an excellent sensitivity with absorbance result. The recoveries of the spiked AFB1in real peanut samples ranged from94.2%to117.3%. Therefore the proposed nano-biosensor was demonstrated to be sensitive, selective, and simple, providing a viable alternative for rapid screening of toxins in agriculture products and foods.(4) Rapid and sensitive aptasensors based on GNR side-by-side and end-to-end assemblies were explored for OTA screening. Aptamer and complementary DNA (cDNA) modified on satellite and end faces of GNRs hybridized under certain conditions, resulting in chain-like structure induced by side-by-side and end-to-end assembly of GNRs. The conformation of aptamer changed in the presence of free OTA molecule in solution, forming a G-quartet of OTA aptamer. As a result, the GNR assemblies reduced with the increasing of OTA concentration, which could been used for OTA detection. GNR side-by-side and end-to-end sassemblies were characterized and optimized. Plasmon coupling of side-by-side GNR assembly was observed stronger than that of end-to-end assembly, which was potential for OTA detecton. Thus, satellite-modified GNR-aptamer and GNR-cDNA probes mixed well and reacted with different concentration of OTA standard solution (0-100ng/mL), and the absorption intensity of GNRs at706nm could be employed as signal indicator for quantification of OTA. Other five mycotoxins common in agro-products were detected for evaluation the selectivity of the proposed aptasensor. It was indicated that the optical aptasensor based on GNR side-by-side assemblies could been potential for OTA discrimination with high sensitivity and specificity.(5) A portable and rapid detecting platform for mycotoxin was constructed, and applied for determination of AFB1content in peanut samples. The relationship between AFB1concentration and absorption intensity of UV-vis spectra at709nm was set up using the platform based on competitive dispersion of GNRs. AFB1in standard PBS solution could be detected linearly from0.22to5.0ng/mL, with a low limit of detection of0.066ng/mL. AFBl in fresh and stored peanut samples purchased from local agricultural market were detected, resulting in ideal spiked recoveries of97.6%and95.7%, respectively. Additionally, the measurement results kept consistent with standard method of HPLC results. Therefore it was indicated that the novel optical nano biosensor based on the portable platform was promising for on-site detection of mycotoxins, with advantages of accurate, reliable and fast measurements, cost-effective, small-size and easy to carry.