A Homogenous Immunosensor For Aflatoxin B₁ Detection Based On FRET Between Different-Sized Quantum Dots

Fluorescence resonance energy transfer(FRET), as a rapid, sensitive and simple analytical protocol, which can be employed for sensing the nanometer-scale changes in donor-acceptor separation distance. Conventionally, the FRET-based system using fluorescence proteins or organic dyes as FRET probes. Such fluorescent sensors are difficult to be excellent spectral overlap and still suffer from narrow absorption spectra, red-tailed emission spectra. In addition, the traditional fluorescence molecules are low chemical stability and susceptibility to photobleaching and interference, and thus, those fundamental properties limited the application of FRET.Quantum dots(QDs) have emerged as promising fluorescent marker, due to their unique photophysical properties, including high quantum yield, broad absorption spectra, narrow and symmetric emission bands, and high resistance to photobleaching and chemical degradation. Therefore, the QD-based FRET analytical method has been widely used in the field of immunoassay, biomolecular binding assay and biosensor. QDs often used as energy donor in the system of FRET, there is few reports that exploit two different emission QDs as the donor and acceptor, respectively. In principle, QDs should be excellent FRET acceptors due to their large absorption cross-section, which can be an order of magnitude larger than that of organic dyes.Aflatoxin B1(AFB1) is the most toxic, mutagenic, teratogenic, carcinogenic secondary metabolites produced by Aspergillus flavus and A. parasiticus. The intake of foodstuff contaminated by AFB1, even at very low of concentration, may be highly dangerous to human health and even cause death. Establishing a rapid, sensitive, and reliable method for sensing the trace level of AFB1 shows great significance to the food safety and monitors the major health problem. Owing its simplicity and utility under homogenous conditions, FRET has been regarded as a powerful tool for investigating individual biomolecules as part of fluorescent immunoassay.Herein, a FRET-based homogeneous immunosensor was developed for sensitive detecting trace amount of AFB1 in rice grains. The assay was consisted of AFB1 labeled green QDs as donor and anti-AFB1 monoclonal antibody labeled red QDs as acceptor. It is based on the concept that the bio-affinity between antigen and antibody made two different sized QDs close enough, thus the FRET occurred. In this work, we propose that the labeling ratio of mAb/antigen onto the surface of QDs is the key factor to achieve a sensitive and reasonable FRET between two different sized QDs.The specific research details of this topic are as follows:Firstly, the monovalent monoclonal antibody(mAb)-labeled red QDs and multivalent hapten-labeled green QDs were designed as energy acceptor and donor, respectively. The anti-AFB1 mAb-labeled red QD was prepared as EDC/NHSS protocol, and green QDs were covalently coupled with oximated AFB1 using a DCC/NHS protocol. Agarose gel electrophoresis and FT-IR spectrum confirm that antibody or antigen was successfully coated onto the surface of QDs, respectively. The QDs-conjugates showed 6%-8% fluorescence weakening, whereas the maximum emission wavelength did not shift compared with the original QDs. The labeling ratios of mAbs to red QD and AFB1 to green QDs were investigated, and the experiment results showed that multivalent AFB1 labeled green QDs and monovalent mAb conjugated red QDs were designed as FRET donor-acceptor pair. The optimal labeling ratio of red QDs to mAbs is 1:10, and the green QDs to AFB1 is 1:10. The reaction ratio of 1:5 between donor and acceptor was selected as the optimal FRET system.Secondly, various buffer parameters including pH value, buffer ionic strength, methanol content, and immunoreaction time were systematically investigated. The buffer conditions(pH, ionic, methanol), has a great influence on the overall FRRT efficiency. The environmental conditions not only affect the immunological reaction, but also may influence the energy transfer between QDs. Under optimization, this study chose the pH 7.4 PB containing 10 mM NaCl and 10% methanol(v/v) at 37℃for 20 min. The linear equation could be represented by Y =-4.065 Ln(x) + 12.142, R2=0.9904, within the range 0.06-5.0 ng/mL. The detection limit of the developed immunoassay was determined to be 0.04 ng/m L. The specificity of the FRET-based assay was evaluated and showed low cross-reaction with other competitors. The recovery rate of spiked samples were in the range 81.28%–97.36%, and the coefficient of variation(CV) in intra-assay and inter-assay was founded within the range 3.9%–5.9% and 7.2%–8.4%, respectively. Statistical analysis using t-test had no significant difference between the proposed FRET-based immunoassay and the commercial enzyme-linked immunosorbent assay kit.