The Study Of Immunosensor For Detection Of Okadaic Acid Of Diarrhetic Shellfish Poisoning

Red tide often happened around the world with the deterioration of the environment.The resultant marine algae would produce a variety of marine biological toxins,including diarrhetic,paralytic and neurologic shellfish poisoning.Among them,through the animal experiment,it proved that diarrhetic shellfish poisoning(DSP)had potential carcinogenicity,teratogenicity and mutagenicity.In the short term there would some symptoms like diarrhea,vomiting and dizziness,et al.which had a great threat to human health.Therefore,it is urgent to develop a rapid,accurate and sensitive method to detect the okadaic acid,one of the DSP.In this paper,we first gave an brief introduction of DSP and the detection methods of DSP.And then,the application of biotin-avidin system and gold nanoparticles in the immunosensor was presented.After that,three kinds of immunosensors were developed Firstly,the traditional ELISA principle was applied to develop kit for detection of OA.Secondly,the biotin-avidin system was introduced into traditional immnosensor for signal amplification.Thirdly,the gold nanoparticles and cascade amplification based on biotin-avidin system and compound of antigen horseradish peroxidase and its antibody were applicated to build a more sensitive immunosensor.These three kinds of sensors could all be used in the detection of OA.The contents of this paper are as follows:1.A kit for rapid detection of okadaic acid on site was developed,and design of the instrument based on the kit for on site detection was completed.Based on competitive immunoreaction,A kit for detecting okadaic acid(OA)was designed.The limit of detection(LOD)is 0.19 ?g/L,and the linear range was 0.2-10?g/L.The cross reaction to Dinophysistoxin-1(DTX1)was 23.6%.The cross reaction to other toxins were almost 0.Spiking experiments showed good recovery rate.The kit has good anti-interference ability to metal ions.At present,the shelf life of the kit was 6 months.The total detection time was within 90min.What's more,the cost is the 1/30 of the Beacon kit.Based on the developed kit,the optical instruments,Bionic e-Eye for detecting OA on site was designed for the screening rapidly.The concentration could be directly displayed on the screen of the phone.The results of real samples detection were in great agreement with the High Performance Liquid Chromatography-Mass Spectromrtry(HPLC-MS)and microplate reader.2.The further immunosensor based on biotin-avidin principle to improve the sensitivity for detection of OA was proposed.On the basis of the kit,the signal amplification biosensor was designed by introducing biotin-avidin system.The antigen of the standard or sample would combine with antibody competitively.The LOD of the proposed biosensor was 0.04?g/L,and the linear range was 0.01-10 ?g/L.Compared to the kit,the LOD of the sensor reduced about 5 times,and the lower limit of the linear range reduced about 20 times.In addition,the cross reaction rate of the sensor to DTX1 was 44.14%,and almost 0 to other toxins.Spiking experimental results showed that recovery rate of the sensor for detecting single OA was in the range of about 91.09%-111.86%and coefficient variation values were less than 15%.Recovery rate of mixed OA and DTX1 was in range of 81.98-132.61%.All above,the sensor had good accuracy.3.A cascade amplification immunosensor based on nano gold and biotin-avidin system was proposed to detect the ultra low concentration of OA.Nano gold had a large surface area and high affinity with biological molecules.Therefore,the signal was amplified to a large degree by combining of secondary antibody-horse radish peroxidase(SAb-HRP)and HRP on gold surface.In addition,by inducing biotin-avidin system and immune complexes of HRP,cascade amplification immunosensor was proposed.the LOD of the sensor is 0.0025 ?g/L,and the linear range was 0.002-5 ?g/L.The lower limit of linear range was 100 times lower than that of the kit.The cross reaction rate to DTX1 was 19.79%,and almost 0 to other toxins.Also,the spiking experiment showed the recovery rate of the sensor was between 76.07%-94.63%,and the CV were less than 15%,which achieved extremely sensitive detection of OA.