A simple and fast protocol for the synthesis of 2-amino-4-(4-formylphenyl)-4H-chromene-3-carbonitrile to develop an optical immunoassay for the quantification of botulinum neurotoxin type F

Abstract

In this study, a novel optical immunoassay platform was developed by using an optical probe 2-amino-4-(4-formylphenyl)-4H-chromene-3-carbonitrile (AFC), which was synthesized via greener Ultra-sonication method. The structure of the probe was elucidated through Fourier-Transform Infrared Spectroscopy and Nuclear Magnetic Resonance spectroscopy techniques. The physicochemical properties of the probe were analyzed by UV-vis and fluorescence spectroscopies. This AFC probe was attached to the antibody via 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide coupling reactions through the formation of amide linkage between them and then it was successfully applied for the detection of botulinum neurotoxin type F through Twisted Intramolecular Charge Transfer (TICT) quenching mechanisms. Under the optimized conditions, the developed immunoassay has shown a linear range response for botulinum neurotoxin type F from 50 to 500 fg mL⁻¹ and limit of detection is 34.4 fg mL⁻¹. The developed immunoassy was tested for variuos food samples and showed a good agreement with our results. This is the first optical immunoassay for the detection of botulinum neurotoxin type F, and it has a wide range of sensitivity and good selectivity, and it is less time consuming and inexpensive method. Finally, the immunoassay was applied to different food samples analysis.