In Situ Optical Fiber Chemiluminescent Biosensors For The Detection Of Mycotoxins In Cereals

The problems of food safety caused by mycotoxins contamination of food and feed have been widely concerned by countries and regions around the world.Rapid,accurate and sensitive detection of mycotoxins in cereal is a necessary step to effectively prevent mycotoxins contamination and protect human health.Most of the existing detection methods have the disadvantages of requiring expensive and sophisticated instruments and complicated operation,which makes it difficult to realize on-site rapid detection.Optical fiber chemiluminescent biosensor has developed rapidly in recent years.Its main advantages are as follows:1)The optical fiber has excellent light transmission performance,which is convenient to construct optical biosensors;2)Optical fiber can be used as solid phase carrier of biometric molecules and signal transmission element at the same time,improving the efficiency of signal collection and simplifying the construction of instrument;3)The optical fiber is small in size and free from electromagnetic interference,which is conducive to the construction of miniaturized instruments and the realization of on-site remote monitoring.However,the core of optical fiber is made of silicon dioxide.Most traditional optical fiber biosensors use silicon chemistry methods to bind biometric molecules on the surface of the fiber.This process is cumbersome and requires a large number of chemical reagents,which limits the further application of optical fiber chemiluminescent biosensor.In this thesis,a series of in situ optical fiber chemiluminescent biosensors were constructed to simplify the preparation process of optical fiber probes and improve the sensitivity of the sensor,and the practical applications were carried out with mycotoxins in cereals as the target.The details are listed as follows:1.Polydopamine-modified optical fiber chemiluminescent immunosensor for detection of deoxynivalenol in wheatPolydopamine（PDA）is formed by the self-polymerization of dopamine under alkaline conditions.Based on this property,a PDA-modified optical fiber chemiluminescent immunosensor（PDA-OFCI）was constructed.A thin layer of PDA was formed on the surface of the optical fiber.The catechol function of PDA can react with the amino group at the end of the protein molecule by Michael addition reaction,so as to fix the complete antigen and other biological recognition molecules on the surface of the optical fiber probe.The competitive immune reaction was used to detect the small molecular deoxynivalenol（DON）.The horseradish peroxidase in the immune complex can catalyze the chemiluminescent substrate to produce chemiluminescence signal,which was collected by optical fiber and transmitted to the photon counter through total internal reflection for signal readout and linear with the concentration of DON.The limit of detection（LOD）of this method for DON is 0.49 ng/m L,and the linearity is good in the range of 1～1000 ng/m L（R²=0.989）.The recovery of DON in wheat samples was between87.85%and 102.31%,and the relative standard deviation（RSD）was less than 14.75%.The detection results of real samples were consistent with those of HPLC-MS.Compared with the traditional optical fiber biosensor,the constructed PDA-OFCI can covalently couple the biological recognition molecules only by modifying the optical fiber with PDA,reducing the use of a large number of reagents in chemical modification,simplifying the preparation process of the optical fiber probe,and has potential application value in the detection of mycotoxins.2.Polystyrene casing embedded optical fiber chemiluminescent immunosensor for detection of deoxynivalenol in cerealsPolystyrene（PS）can adsorb proteins and other biological recognition molecules on its surface through hydrophobic interaction.In this chapter,a PS casing embedded optical fiber chemiluminescent immunosensor（PS-OFCI）was constructed by combining PS casing and automatic single-channel chemiluminescence analyzer,and was applied to the detection of DON in wheat and maize.The LOD of this method is 0.29 ng/m L,and the linear range is 0.5～2000 ng/m L（R²=0.985）.The recoveries of DON in wheat and maize samples were 96.64%～106.39%and 87.82%～109.4%,and the RSD were less than 8.72%and 10.72%,respectively.The results of real samples were consistent with HPLC-MS.Physical adsorption of PS casing avoids the use of chemical reagents,simplifies the preparation process of optical fiber probe,and combines with automatic analysis equipment to reduce the operating steps of detection personnel,realizing automatic detection of DON.3.Hybridization chain reaction and Clostridium butyricum Argonaute-mediated optical fiber chemiluminescent aptasensor for detection of aflatoxin B₁ in maizeA hybridization chain reaction（HCR）and Clostridium butyricum Argonaute（Cb Ago）-mediated chemiluminescent optical fiber aptasensor was constructed to realize the sensitive detection of aflatoxin B₁（AFB₁）.In this chapter,the PS casing embedded optical fiber probe was used to adsorb streptavidin,and the initiator DNA of HCR was coupled to the surface of the optical fiber through the streptavidin-biotin system.Biotin-H1 and Biotin-H2 were added to initiate the HCR signal amplification system.With AFB₁ aptamer as the biological recognition molecule,guide DNA（g-DNA）and aptamer complement each other to form a duplex structure.When the target existed,aptamer recognized and captured it,and released g-DNA at the same time.After assembly of g-DNA and Cb Ago,initiator DNA can be cleaved to remove HCR amplification products.Horseradish peroxidase labeled streptavidin combines with biotin in the remaining HCR amplification products on the optical fiber,and horseradish peroxidase catalyzes chemiluminescent substrate for signal readout.HCR signal amplification and cleavage activity of Cb Ago improve the sensitivity of the sensor;with programmable cleavage capacity of Cb Ago,the detection of different targets can be realized only by designing corresponding g-DNA and initiator DNA.With AFB₁ as the analyte model,the LOD of this method is 1.31 pg/m L,and the linearity is good in the range of 5～50000 pg/m L（R²=0.990）.The intra-assay and inter-assay recoveries of AFB₁ in maize samples were 81.86%～104.37%and 79.41%～114.8%,and the intra-and inter-RSD were less than 7.21%and 16.35%,respectively.The detection results of real samples were consistent with those of HPLC-MS.