Photonic Crystal Sensing Technique For Highly Sensitive And Label-free Detection Of Staphylococcus Aureus Enterotoxin B In Food

Biotoxins are harmful components produced by animals,plants and microorganisms.It is an important factor affecting food safety and has been listed as an important source of foodborne diseases by the World Health Organization（WHO）.Foodborne diseases related to biotoxins are one of the most prominent public health problems in the world,which directly lead to food poisoning.As one of the superantigens that cause food poisoning in humans,staphylococcus aureus enterotoxin B（SEB）has already been listed in the International Convention on the Prohibition of Chemical Weapons because of the enormous harm they may cause,whose lethal dose is as low as 20 ng kg^-1.At present,methods including nucleic acid amplification,immunoassay,chromatography,and colorimetry are applied in SEB detection.However,these methods often rely on expensive instruments with low sensitivity,which is not suitable on-site detection.Therefore,it is of great significance to develop a highly sensitive and accurate biosensing method for SEB detection.In recent years,photonic crystal（PC）-based biosensors have been widely applied in food safety detection because of their advantages such as no labeling and self-expression of sensing signals.Benefiting from the array structure of large pore,the inverse opal photonic crystals（IOPCs）can provide larger specific surface area and more interaction sites.Herein,taking advantage of the good biocompatibility and flexibility of polymer materials,IOPC with macroporous network interpenetrating structure was used as the biosensing platform,and a new SEB biosensing method was developed.The method is easy to prepare with high specificity and good accuracy.The main contents of this study are as follows:（1）An aptamers（Apt）modified polymer-IOPC biosensing method was developed to achieve highly specific and sensitive detection of SEB.Firstly,opal Si O₂-PC was prepared by vertical sedimentation self-assembly method,forming the preparation template.Methacrylic acid（MAA）and the ethylene glycol dimethacrylate（EGDMA）were selected as the polymerization monomer and the crosslinking agent,respectively,and diisoheptonitrile（ABVN）was added as the photoinitiator to form the prepolymerization solution.The prepolymerization solution was then filled into the cracks of the opal structure template and polymerized under UV light,forming the polymer-coated composite material.After that,the Si O₂ microspheres in the composites were removed by etching with hydrofluoric acid,forming the IOPC material with three-dimensional porous structure.Finally,the amino modified SEB Apt is attached to the surface of the activated polymer IOPC material.The maximum reflected wavelength of the material could be blue-shifted with the increase of SEB concentration,whose maximum reflected wavelength were linearly with the logarithm value of SEB concentration in the range of 10^-2 pg m L^-1 to 10³ pg m L^-1,and the detection limit was as low as 8.7443 fg m L^-1,performing good accuracy in practical detection applications.（2）To further improve the sensitivity of biosensing method,gold nanoparticles（AuNPs） were introduced in the PC.Based on the enhancing local surface plasmon resonance effect（LSPR）,an AuNPs doped colorimetric IOPC material was developed to realize ultra-sensitive and visual detection of SEB.The IOPC material was prepared by etching template cross polymerization of MAA and EGDMA and doped with AuNPs on the polymer backbone.The introduction of AuNPs provided more Apt modification sites and effectively supported the polymer skeleton.Under optimized experimental conditions,the detection method can detect SEB in the range of 10^-2 pg m L^-1 to 10³ pg m L^-1,whose LODs was 4.290 fg m L^-1.Furthermore,such as with p H test paper,SEBs as low as 1 pg m L^-1 could be detected by observing its bright color variation without the need for equipment.In addition,the IOPC biosensing method provides a universal sensor detection platform that can be used to visually detect other hazards by replacing the identification element.In this paper,the SEB detection method was developed by combining the large pore structure of IOPC material and the good flexibility of polymer material,which showed high specificity and high accuracy.Then,the ultra-sensitive and visual SEB detection method was also developed with the introduction of AuNPs.Compared with the SEB detection methods reported before,the AuNPs-IOPC detection method has significant advantages in sensitivity,detection range and operating procedures,which has a potential application prospect in food safety and biosensing.