Fluorescent sensors for biosensing applications: immunosensors for mycotoxins and aptasensors for interferon-gamma
thesisposted on 2022-03-28, 17:14 authored by Fuyuan Zhang
In this thesis, recent advances in the development of fluorescent sensors for the detection of mycotoxins and interferon-gamma (IFN-γ) were summarized. Three CdTe/CdS/ZnS quantum dot (QD)-based fluorescence immunoassays were developed for monitoring two types of mycotoxins: zearalenone (ZEN) and aflatoxin B1 (AFB1), and different fluorescence aptasensing strategies were developed to explore the detection capability of an anti-IFN-γ aptamer. A novel quantitative fluorescence immunoassay (FLISA) and a rapidly responding semi-quantitative fluorescent immunochromatographic test strip (ICTS) were established for the detection of ZEN in maize. The detection limit (LOD) of FLISA was determined to be 0.012 ng/mL in maize, with a dynamic range from 0.038 to 0.977 ng/mL. The rapid ICTS had a visual LOD of 1.5 ng/mL in maize extract. Two novel magnetic nanobeads-based fluoroimmunoassays were designed for sensitive detection of ZEN in cereals. Under optimal conditions, concentration as low as 0.019 ng/mL and 0.049 ng/mL ZEN could be detected in the extract solution for the first capture last react (FCLR) procedure and first react last capture (FRLC) procedure, respectively. The present methods were further performed for detection of ZEN in real cereal samples, and the LODs were 0.6 μg/kg and 1.5 μg/kg in the FCLR and FRLC, respectively. It showed that the proposed fluoroimmunoassays have great potential in bioanalysis. A simple FLISA was developed based on CdTe/CdS/ZnS QD for monitoring AFB1 in cereals. The proposed FLISA displayed a broad detection of AFB1 ranging from 0.08 to 1.97 ng/mL with a low LOD of 0.01 ng/mL. Moreover, the immunoglobulin genes of the anti-AFB1 Fab were cloned, and the recombinant Fab antibody for AFB1was prepared with a Kd value of 1.09×10-7 mol/L. To investigate the interactions between the antibody and AFB1, molecular dynamic simulation and quantum-chemical computation were performed on the Fab-fragment homology model. The analysis results showed that hydrogen bonds formation, Pi-alkyl interactions, and van der Waals interactions played the most important roles in antibody recognition. A strand displacement strategy for IFN-γ was developed by introducing three different complementary strands and two different signal/quencher pairs. The Texas red/BHQ2-based sensor demonstrated the best equilibrium binding constants of 21.87 ng/mL to IFN-γ. To improve the sensitivity of this aptamer-based sensor, a hybridization chain reactions amplification strategy was introduced, yielding a 12-fold improved sensitivity of 0.45 ng/mL. In addition, a fluorescent optical fiber aptasensor was developed for IFN-γ sensoring based on the strand displacement strategy.