HIGHLY SENSITIVE FLUORESCENT APTASENSOR VIA HAIRPIN STRUCTURE OF G-QUADRUPLEX OLIGONUCLEOTIDE-APTAMER CHIMERA AND SILICA NANOPARTICLES FOR AFLATOXIN B1 DETECTION IN THE GRAPE JUICE AND HUMAN SERUM SAMPLES

Background and Aim: Among the aflatoxins, aflatoxin B1 (AFB1) is a major food pollutant which can pose a high risk to human health and has been listed as group 1 carcinogen as announced by the International Agency for Research on Cancer(1, 2). This mycotoxin is resistant to high temperature and can occur in various food materials and agriculture commodities, such as grains, peanuts and fruits(3, 4). So, the development of reliable and sensitive analytical techniques to monitor levels of AFB1 in food products is required to avoid exposure to AFB1.Aptamers are single-chain nucleic acids with good selective recognition potentials toward various types of targets, including biological macromolecules and cells. They are obtained from randomized nucleic acid libraries via an in vitro procedure called as SELEX (Systematic Evolution of Ligands by Exponential enrichment)(5, 6).Among different analytical strategies, aptamer-based fluorescent sensors are especially attractive, owing to their fast response, small reaction volumes, easy readout and good sensitivity(7). Silica nanoparticles (SNPs) can act as fluorescent enhancers of fluorophores (8-10). In this work, an accurate fluorescent sensing method was proposed for AFB1 determination, based on hairpin structure of G-quadruplex oligonucleotide-Aptamer chimera, silica nanoparticles coated with streptavidin and N-methyl mesoporphyrin IX (NMM).Methods: In the absence of target, the G-quadruplex forming sequence was locked in the hairpin structure of chimera. So, upon addition of NMM, little amounts of the fluorophore could bind to the SNPs-Streptavidin through hairpin structure of chimera and thus, a weak fluorescence signal was recorded. Upon the introduction of AFB1 and NMM to the Chimera-modified SNPs-Streptavidin complex, the formed Aptamer/AFB1 complex led to disassembly of the hairpin structure of chimera and formation of G quadruplex structure. Therefore, huge amounts of NMM bound to G quadruplex structure on the surface of SNPs-Streptavidin, resulting in a strong fluorescence signal. Moreover, binding of the NMM to SNPs-Streptavidin via G-quadruplex structure changed the environment of the fluorophore and its fluorescence intensity was more amplified by SNPs-Streptavidin. At first Optimization of SNPs-streptavidin concentration was done. then, 50 µL Chimera-modified SNPs-Streptavidin complex (50 nM chimera) in 10 mM PBS (pH 7.4) was treated with 100 µL PBS (10 mM PBS, pH 7.4) containing various concentrations of AFB1 (0–1500 pg/mL) and 4 µL NMM (2 µM) for 30 min at 37 °C (final sample volume 154 µL).Then, fluorescence intensities were obtained with emission wavelength at 610 nm and excitation at 399 nm. Specificity of the proposed aptasensor for AFB1 was checked. Moreover, to verify the analytical performance of the proposed aptasensor in real samples, serum and grape juice samples containing different concentrations of AFB1 were analyzed for AFB1 detection using the presented aptasensor.Conclusion: The hairpin structure of chimera and SNPs-Streptavidin allowed AFB1 detection with high sensitivity and specificity. Furthermore, the developed sensor could detect AFB1 in 30 min. The relative fluorescence intensity increased as AFB1 concentrations increased with a linear range of 30–900 pg/mL and a limit of detection (LOD) of 8 pg/mL. The constructed aptasensor was successfully employed to assess AFB1 spiked grape juice and human serum samples. The analytical recovery of AFB1 in the grape juice samples ranged from 95 to 106%, implying the great potential of the presented aptasensor in food product analysis.

Keywords:

Fluorescent aptasensor , Chimera , Aflatoxin B1 , N-Methyl mesoporphyrin IX , Silica nanoparticles