Development Of Electrochemical Biosensor For Detection Of Fumonisin B₁

This study was aimed to establish three methods for the detection of FB1 using electrochemical biosensors.The first method was based on the nano composites of GS-Au,and the other one was based on GS-TH.And the electrochemical biosensors was compared with ELISA.1 Construction of electrochemical aptamer sensor based on GS-THIn this study,a novel aptasensor based on grapheme-thionine nanocomposites(GS-TH)was established for sensitive detection of FB1.Initially,GS was sonic disrupted and combined with TH.Gold nanoparticles were prepared by improved HAuCl4 reduction method.The diameter of Au nanoparticles were 13 nm,through the observation in a transmission electron microscopy.The working electrode was glassy carbon electrode.Gold nanoparticles were dried on the electrode surface and modified with capture DNA(DNA1),which bound to aptamer DNA specially.The electrode modified was immersed into different concentration of FB1 and DNA2 which was completely complemented to DNA1.Some DNAA released from electrode surface when FB1 specifically bind to DNA aptamers.And DNA2 could combined with DNA1.Then the electrode was dropped into GS-TH solution.GS-TH then bound to the single part of DNAA.The signal of TH on the surface of electrode was detected by cyclic voltammetry to quantify FB1.The detection conditions were optimized.The best concentration of DNAA for reaction was 0.5 μM,with 3 h for DNAA and GS-TH fixation.The electrochemical aptamer sensor was constructed to creat the standard curve of FB1,and the concentration of FB1 in a linear ranged from 10-11 to 10-4 mg/mL.The equation was I(μA)=1.37997-0.15084×lgCFB1(g/mL),with a correlation coefficient of 0.9945.The limit determination contration of FB1 was 10 pg/mL.The results showed that the aptasensor was repeatable,reproducible,stable and specific.2 Construction of electrochemical aptamer sensor based on GS-AuIn this work,a novel aptasensor based on grapheme-gold nanoparticles nanocomposites(GS-Au)was established to detect FB1.Firstly,we prepared GS-Au and the nanocomposites were observed by transmission electron microscopy characterization.It was found that the gold nanoparticles spaced at regulair on GS.The working electrode was glassy carbon.GS-Au were dried on the surface of electrode and modified with aptamer DNA.The glassy carbon electrodes was dropped into FB1 solution of different concentration.FB1 combined to the aptamer DNA and resulted in decreased electrochemical signal.FB1 was quantified by cyclic voltammentry detection of the surface reduction of current value.The determination conditions were optimized.The best concentration of DNAA for reaction was 0.1 μM,with 1 h for the optimal fixation.Under the optimal conditions,the electrochemical aptamer sensor was constructed to creat the standard curve of FB1,and the concentration of FB1 in a linear ranged from 10-11 to 10-4 g/mL.The equation was ΔI(μA)=1.064+0.089×lgCFB1(g/mL),with a correlation coefficient of 0.9965.The limit determination contration of FB1 was 10 pg/mL.The results showed that the aptasensor was repeatable,reproducible,stable and specific.3 Colorimetric aptasensor for fumonisin b1 detection by regulating the amount of bubbles in closed bipolar platformA novel electrochromic biosensor has been developed by coupling the chemical sensing reactions at closed bipolar electrode(BPE)to the electrochemical reactions at the driving electrode for colorimetric detection of fumonisin B1(FB1).In order to achieve FB1 detection,a recognition probe was prepared by conjugating Au nanoparticles(NPs)labeled FB1 aptamer to capture DNA-Fe3O4 NPs surface.After the chemical reduction of silver ion on Au NPs,the prepared probes were introduced to GCE surface to catalyze the decomposition of H2O2 to oxygen bubbles by silver particles.The accumulation of numerous bubbles led to a significant decrement of the effective area of GCE cathode and current flow through the electric bridge which was reported by the inhibited electro-deposition of prussian blue(PB)at ITO driving electrode in sensing cell.After adding FB1,Au NPs-aptamer was liberated from Fe3O4 NPs-capture DNA surface,resulting in a decreased amount of silver particles assembled on GCE and an increased amount of PB deposited at ITO electrode.The linear relationship between ΔIRed and the logarithmic values of FB1 concentration was 10-9 to 10-5 mg/mL.The linear equation wasΔIRed=-98.47-9.8 logC(mg/mL),and the correlation coefficient was 0.9987.This indirect colorimetric strategy provides a simple,repeatable and portable for bioanalysis.4 Comparison of electrochemical aptasensor with ELISAIn this study,the ELISA method was established.FB1 and carrier proteins ovalbumin(OVA)were synthesized to FB1-OVA.Through the ascites preparation methods,the monoclonal antibodies of FB1 were prepared.Firstly,to recover th hybridoma cells,then injected intraperitoneally to 20 female Balb/c mice.During the experiment the mice were observed daily.When the mice showed swollen abdomen,the ascites were collected and purified with protein purification column of HiTrap Protein G HP.The monoclonal antibodies were obtained.By SDS-PAGE evaluation,FB1-OVA and FB1 antibody were found synthetised successfully.The standard curve of ELISA was created.The detection contration of FB1 range from 0.1 ng/mL to 1000 ng/mL,with a linear correlation coefficient of 0.9895.The limit detection concentration was 1.38 ng/mL.The electrochemical aptamer sensor standard curve was established in test 1.the two methods wer both repeatable,reproducible,stable and specific.Compared with ELISA,electrochemical aptamer sensor has a wide detection range and low detection limit.It coule be easily contructed,the aptamer has higher stability and cheaper than antibody.