Development And Application Of Electrochemical Biosensor For Detection Of Fumonisin B₁ In Feedstuffs

Mycotoxins are a series of toxic secondary metabolites produced by various fungus.In different kinds of mycotoxins,fumonisins are the most common mycotoxins in maize-growing areas in the world.Due to its relationship with human and animal diseases,fumonisins are considered as a serious issue in maize and grain.Fumonisins,including fumonisins B₁,B₂ and B₃,are naturally producted by Fusarium verticillioides,Fusarium proliferatum,and Gibberella fujikuroi.Fumonisins can cause a serious and even fatal equine leukoencephalomalacia disease in horses,or fatal porcine pulmonary edema disease in pigs.It is considered as a kind of possible human carcinogens by International Agency for Research on Cancer（IARC）.Recently,the detection technology of mycotoxin has been developed very well,for instance,thin layer chromatography（TLC）,high performance liquid chromatography（HPLC）,high performance liquid chromatography-mass spectrometry（HPLC-MS）and enzyme linked immunosorbent assay（ELISA）,etc.Currently,HPLC is the most commonly used method because of its higher sensitivity and reliability.However,the samples have to be extracted and purified which is very complicated and time consuming.Electrochemical biosensors,combining with biological analysis method and electrochemical sensor technology,are highly selective and sensitive,and also have very broad application prospects in bioengineering,environmental monitoring,pharmaceutical industry,food safety,agricultural analysis,etc.Nano materials have been widely applied in electrochemical biosensors with its advantage of large specific surface area,high catalytic activity,strong affinity and excellent biological compatibility,etc.In recent years,the electrochemical biosensor technology,with its miniaturized,high sensibility and strong specificity advantage,has become a rapid detection tool for mycotoxin.In this study,we prepared Fumonisin B₁ complete antigens and its monoclonal antibody.We used indirect and direct competitive methods,and thione as the signal probe to construct Fumonisin B₁ biosensor.Test I Construction of electrochemical immunosensor based on the signal amplification of magnetic nanoparticles and graphene.To prepare FB₁ monoclonal antibody and evaluate its titers,glutaraldehyde method was applied to couple FB₁ with carrier proteins ovalbumin（OVA）and make synthesized FB₁-OVA.The protein concentration of FB₁-OVA was accessed by Coomassie brilliant blue kit as 1 mg/mL.The complete antigen FB₁-OVA was successfully conjugated and tested by SDS-PAGE.Then FB₁-OVA was used as a coating antigen in detection of antibody titer by ELISA.FB₁ monoclonal antibodies were prepared by ascites preparation techniques.First,hybridoma cells were recovered and observed their cell morphology.The antibody titer and stability were tested in supernatant.The recovered hybridoma cells were in good condition. The monoclonal antibody titers in supernatant were 1:1600,with good antibody secreting stability.Then,20 female Balb/c mice were selected and injected hybridoma cells suspension intraperitoneally.During the study,mice were monitored every day.Ascites were collected when the mice had swollen abdomen.The ascites were purified with HiTrap Protein G HP protein purification column.The protein concentration of FB₁ monoclonal antibody was assessed as 10 mg/mL.Through SDS-PAGE evaluation,the molecular weight of FB₁ monoclonal antibody was 149.50 kDa with higher purity.The FB₁ monoclonal antibody titers was 1:102400 by ELISA which can be used to construct the electrochemical immunosensor.In this study,thionine was used as electroactive probe to construct a new kind of Fe3O4magnetic beads,which were then applied as a carrier signal molecular for the construction of a novel electrochemical immunosensor for the detection of FB₁.First,the graphene thionine nanocomplex was synthesized with thionine and graphene,which was successfully detected by UV spectrophotometry.And then,Fe3O4 beads/Ab2 conjugates were obtained by coupling with Fe3O4 beads and sheep anti-rat antibody by glutaraldehyde.Graphene thionine nanocompounds were activated by EDC and NHS,which were connected with amino in Fe3O4 beads/Ab2 conjugates and then turned into the signal molecules of the electrochemical immunosensor.In this study,gold electrode was used as the working electrode.Gold electrode was immersed into cysteine for self-assembly.Glutaraldehyde was applied as a connect arm to modify FB₁-BSA conjugates.The nonspecific binding sites were blocked by 3%BSA solution.FB₁ of the sample solution competitively combined to Ab1 with surface fixed FB₁-BSA conjugates on the electrode.The signal molecules were connected to Ab1.Cyclic voltammetric was applied to measure the current values of electrode surface thionine and quantify FB₁.In this study,Electrochemical Impedance Spectroscop was used to characterize the electrode modification process.It turned out that the electrode surface was assembled layer by layer,and the electron transfer obstacles were gradually increased.Detection conditions of electrochemical immunosensor were optimized. We found that the optimal concentration of coating antigen FB₁-BSA was 100 g/mL,the optimum dilution ratio of Ab1 was 1:7000,the optimum dilution ratio of Ab2 was 1:10000,the optimal pH of cyclic voltammetry detection solution was 7.0,the optimal incubation time of FB₁ competitively binding with FB₁-BSA to Ab1 was 40 min,and the reaction temperature was 37℃.Under the optimum conditions,the linear range of the immunosensor was from 10-8 mg/mL to 10-4 mg/mL,the linear equation was I（μA）=55.7-66.2×1gCFB₁（mg/mL）,the correlation coefficient was-0.9989,and the detection limit was 0.01 ng/mL.These results indicated that this immunosensor has good repeatability,reproducibility,stability and specificity.Test II Construction of electrochemical aptasensor based on the dual signal amplification of Au nanoparticles and graphene thionine nanocomposites.In this study,a novel aptasensor with the dual amplification of Au nanoparticles（AuNPs）and graphene/thionine nanocomposites（GS-TH）were constructed for sensitive determination of FB₁.Initially,we prepared gold nanoparticles by improved HAuCl4reduction method.By transmission electron microscopy charaterization,the diameter of gold nanoparticles were 13 nm.The glassy carbon electrode was used as working electrode. Gold nanoparticles were dropped onto the electrode surface to dry and then modified with capture DNA.The captured DNA specifically bound to aptamer DNA.The modified electrode was immersed into graphene thionine solution for self-assembly.The electrochemical signal of probe thionine was detected by cyclic voltammetry.After specific binding of FB₁ and DNA aptamers,the GS-TH was released from electrode surface,which resulted in decreased electrochemical signal.The surface reduction of thionine was detected by cyclic voltammetry to quantify FB₁.The detection conditions of electrochemical aptamer sensor were optimized.The optimal reaction concentration of capture DNA was0.25μM,and the best fixation time of graphene thionine was 3 h.Under these optimum conditions,we established the electrochemical aptamer sensor to constract FB₁ standard curve,and the linear in the range of FB₁ concentration is from 10-12 to 10-4 mg/mL,the linear equation was△I（μA）=1.175+0.067×1gCFB₁（mg/mL）,the correlation coefficient was 0.9982,and the detection limit of FB₁ was 1 pg/mL.These results indicated that this aptasensor has good repeatability,reproducibility,stability and specificity.Test Ⅲ Comparison of the detecting methods between electrochemical aptasensor and HPLC The electrochemical aptamer sensor standard curve was established in testⅡ.The detection limit of FB₁ was 1μg/kg through spiked sample detection.The recovery rate of FB₁ from maize,wheat and complete feed samples was measured,when we added the concentration of FB₁ was 5μg/kg,50μg/kg and 100μg/kg,respectively.The results showed the recovery rate of these three kinds of feed was higher than 90.0%,and the variation coefficient was less than 10.0%.In this study,the HPLC standard curve was established.The detection range of FB₁was 10～1000 ng/mL,and the linear correlation coefficient was 0.9998.The detection limit of FB₁ was 10μg/kg by spiked sample detection.In addition,the recovery rate of FB₁ from maize,wheat and complete feed samples was measured.The concentrations of added FB₁samples were 50μg/kg,100μg/kg and 200μg/kg,respectively.The results showed that the recovery rate of three kinds of feed was higher than 80.0%,and the variation coefficient was less than 10.0%.62 feed samples were collected from all over the country,including maize,wheat,complete feed,etc.Electrochemical aptasensor and HPLC were applied to detect the concentration of FB₁ in those extracted and purified samples.It turned out that the detection rate of two methods were all 100%.The average content of FB₁ in the sample was 833.3μg/kg by electrochemical aptasensor,which was higher than the value of 791.44μg/kg in HPLC detection.The correlation of the two methods for detecting FB₁ in feed were measured.To establish the correlation curve,HPLC detection results and the aptamer sensor results from the same sample were set up as abscissa and ordinate,respectively.The correlation coefficient was shown as 0.9999.These results demonstrated that the aptamer sensor and HPLC methods have a good correlation in detecting of FB₁ in feed,indicating that during the linear range,the electrochemical aptamsensor can be used for detection of FB₁ in agricultural products with higher sensitivity than HPLC.