| Corn is one of the three largest grains in the world,not only an important food product,but also an important component of many industrial raw materials.China,as a major producer and consumer of corn,the quality and safety of corn is closely related to the dietary safety of consumers and the sustainable development of the grain consumption market.However,corn is susceptible to contamination by various mycotoxins during production,storage,and transportation,resulting in the loss of food resources and affecting the competitiveness of corn trade exports in the international market.Currently,conventional detection methods used to detect mycotoxins in food crops have limitations such as cumbersome detection steps,high detection costs and long detection cycle.Moreover,the equipment is too large to meet the requirements for rapid detection of test samples in situ.Recently,surface enhanced Raman spectroscopy(SERS)detection technology has attracted more and more scientific researchers’ attention due to its narrow peak shape,high resolution fingerprint characteristics,and its spectra can provide information such as the structure and conformation of sample molecules.It has been widely applied in fields such as biomedicine,material analysis,medical imaging and food safety testing.In this study,two typical mycotoxins in corn,zearalenone(ZEN)and ochratoxin(OTA)were used as research objects.SERS technology combining with the preparation of metal and metal coupling materials,functionalization of biological aptamers and specific molecular recognition to develop the SERS detection methods with strong specificity and high sensitivity.SERS aptasensor detection systems for the trace detection of ZEN,OTA and the simultaneous detection to the above two mycotoxins in corn has been implemented.This study is of great significance for timely monitoring the mycotoxins pollution in grain,ensuring the quality and safety of grain and promoting the healthy development of grain industry.The specific research content is as follows:1.SERS detection of zearalenone by gated molecular aptamers.ZEN is a nonsteroidal estrogen mycotoxin,originally found in moldy corn,which can cause serious damage to the immune,endocrine and reproductive systems of humans and animals.This study developed a specific SERS aptasensor technique for ZEN detection in corn.1)The aminated mesoporous silica adsorbed the Raman signal molecule Rhodamine 6G(Rh6G)and further grown gold nanoparticles(Au NPs)on its surface through gold seed growth.2)The hot spots generated between Au NPs and mesoporous gaps greatly amplified the Raman signal of Rh6 G.3)After ZEN aptamers further functionalizing of the Au NPs on the surface of mesoporous silicon,the spatial and electrostatic effected of the aptamers produce masking effects on the Raman signal response of Rh6 G.After recognizing and binding of the aptamers to the ZEN,changes in aptamers spatial structure weakening the signal masking effect,and the Raman intensity of Rh6 G undergoes varying degrees of recovery.Based on this,a linear quantitative relationship between SERS characteristic peak intensity of Rh6 G and ZEN content was established.4)Exploring the optimal experimental conditions to establish a SERS specific detection system for the recognition of ZEN and to analyze the specificity of SERS method and the differences between this method and traditional detection methods.The SERS aptasensor constructed by our research is suitable for ZEN within the range of 6-400 μg/kg,exhibiting good selectivity with a detection limit of 0.0128 μg/kg which could be used for rapid detection of ZEN in corn.2.SERS detection of ochratoxin based on dendritic magnetic silver composite structure bonded alloy nanoparticle assemblies.OTA is a toxic metabolite produced by Penicillium and Aspergillus and is widely found in cereals and other agricultural products,intaking excessively would cause irreversible damage to the kidneys and liver.In this study,a SERS detection technique for the specific recognition of OTA by an assembly of magnetic silver composite dendritic structures and functionalized aptamers was developed.1)Magnetic nanospheres were used as the synthetic substrate,further,dendritic silver shells were grown by sonochemical synthesis to adsorb Raman signal molecules 4-ATP,and OTA aptamers were finally modified as capture probes and SERS enhancement substrates.2)Au NPs was functionalized with OTA aptamer complementary chains as Raman enhancement probes,and the enhancement substrate and probes are assembled into SERS aptasensor components through complementary interactions between the aptamer chains.3)Nano gap between the enhancement substrate with dendritic structure and Au NPs allowing the SERS aptasensor exhibiting strong Raman intensity.Presence of OTA caused the key regions of the OTA aptamers on the SERS enhancement substrate to selectively bind to OTA,leading to the dissociation of the Raman enhancement probes from the SERS enhancement substrate.After the magnetic separation,the Raman intensity is significantly reduced.4)Optimizing the condition parameters of key experimental steps,SERS aptasensor was used to collect SERS spectra for gradient concentration OTA standard sample solutions,and to establish standard curves.This SERS aptasensor detection method can achieve micro detection of OTA ranging from 0.005 to 75 μg/kg with a minimum detection limit of 0.0312 μg/kg.This indicated that SERS aptasensor constructed by the enhanced substrate with metal coupling property and combined with the aptamer functionalization can be used for rapid detection of OTA in corn.3.Simultaneous SERS detection of OTA and ZEN based on rigid SERS reinforced substrate.In practice,corn is usually infected by more than one mycotoxin,which increases the difficulty of detecting mycotoxins in corn.Based on conductive glass,a rigid material,through electrochemical deposition and the differential characteristic peaks between two Raman signal moleculars,this study developed a rigid and portable SERS quantitative detection method capable of simultaneously detecting two mycotoxins OTA and ZEN.1)Layer of Au NPs was deposited on the surface of conductive glass(ITO)by electrodeposition,and then graphene oxide(GO)was coated to prepare a SERS enhancement substrate.2)Raman signal probes with gold core silver shell structures functionalized by OTA and ZEN aptamers were prepared,and Raman signal molecules 4-mercaptopyridine(4-MPy)and 4-mercaptobenzonitrile(TBN)with significantly different characteristic peaks were adsorbed on the shells of the two signal probes.3)Through a non covalent interaction(π-π bond)between the signal probes and graphene oxide on the ITO/Au NPs/GO surface,the signal probes were adsorbed on the rigid reinforced substrate surface,resulting in a SERS enhancement effect with two characteristic peak signals.When a rigid SERS enhancement substrate is immersed in a solution with a detection targets,the preferential and specific binding between the detection target and the aptamer made the signal probes dissociated from the surface of the enhancement substrate,resulting in varying degrees of reduction in SERS signal intensity.4)Experimental conditions were optimized,and the SERS spectra was collected for OTA and ZEN standard samples with gradient concentration.The standard curve was established according to the signal intensity of corresponding characteristic peaks,and the specificity and spiked recovery rate of the detection method were analyzed.The double mycotoxin SERS aptasensor detection method explored in this study was applied to detect OTA ranging 0.002-40 μg/kg with the limit detection of 0.476 μg/kg,for content of ZEN ranging from 0.2 to 200 μg/kg,The minimum detection limit was 0.374 μg/kg.This dual mycotoxin simultaneous detection by SERS method shows great potential in the rigid enhancement substrate and simultaneous detection of multiple mycotoxins.Aiming at two common mycotoxins in corn,ZEN and OTA,three different SERS aptasensors were constructed based on SERS spectra detection technology combined with biological aptamer,which realized the detection of a single mycotoxins and the mixed detection of two mycotoxins.Various types of SERS enhancement substrates such as metal coupling materials,magnetic coupling materials and rigid substrates,multiple types of Raman signal molecules and functionalization of nano material by aptamers were used,which provided the research ideas for the subsequent SERS detection technology for a variety of mycotoxins. |
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