The Preparation And Application Of Poly-(3-hydroxy-Butyrate-co-3-hydroxyvalerate)Microspheres As A New Carrier In Immunization Detection

At present,most of the carriers used in immunoassay are made of non-degradable polymer materials.As a large-scale of immune detection is used in the future,more and more carriers will be used in immunoassay,and become waste.Improper discard of this waste will cause environmental pollution.Therefore,how to find degradable materials as carriers in immunoassay becomes very meaningful.In this work,the biodegradable poly(3-hydroxybutyrate-co-3-hydroxyvalerate)(PHBV)was used as the material to prepare PHBV microspheres with the multiple emulsion method.And the technique parameters of PHBV microsphere preparation were optimized too.The PHBV microspheres were used as the carrier to detect fibronection(FN)for the first time.In order to simplify the complicated centrifugal process,magnetic PHBV microspheres were prepared with adding Fe3O4 nano particles,and used as the carrier to detect FN.The experimental results showed that the immunoassay process could be greatly simplified with the magnetic separation,and the detection time could be shortened much.In the study of PHBV microsphere preparation with multiple emulsion method,the property of the PHBV microspheres were characterized by microscopic examination,scanning electron microscope(SEM),and conductometric titration for determining the carboxyl content in the surface of PHBV microspheres.The influences of the solvent concentration,emulgent concentration,emulsification rotation speed,oil water proportional were studied.Based on the microspheres diameter,uniformity of size,and dispersity,the optimal conditions for preparing PHBV microspheres were found as follows,the PHBVconcentration is 5%w/v,the PVA concentration in W2 1%w/v,the secondary emulsion speed 1500 rpm,the W1/O 1:10,the(W1/O)/W2 1:10.The PHBV microspheres prepared in the optimal conditions have a good dispersibility,and the diameter of PHBV microspheres are around 5?15 ?m.The carboxyl content on the surface could reach 3.0mmol/g.When the prepared PHBV microspheres were used as the carrier in FN detection,the optimal concetration of different reagents were found as follows,the amount of the PHBV microspheres was 4 milligram,goat anti human FN antibody with 1:32 potency,enzyme-labeled antibody with 2000 dilution.Based on the above conditions,it was found that the lowest detection limitation of FN is 1?g/ml,the highest detection limitation 100?ug/ml.Using this method to detect the FN concentration in the serum and standard samples,the detection results obtained from the PHBV microspheres carrier are almost identical with that from ELISA plate.The time spent on detection by ELISA plate was more than 6.5 hours,however,3.5 hours was enough for PHBV microspheres.So it was confirmed that the PHBV microspheres can be used as the carrier to detect the FN concentration,and the detection time in this method was shortened much.In order to further simplify the FN detection process with PHBV as carrier,the magnetic PHBV microspheres were prepared with adding the Fe3O4 nano particles,and used for FN detection.The experimental results showed that the optimal conditions for preparing magnetic PHBV microspheres are as follows,the additive amount of the Fe3O4 nano particles was 100mg,the PVA concentration 3%w/v,the reaction time 4 hours.Based on the optimal conditions,the magnetic PHBV microspheres with good dispersibility and good magnetic performance were prepared,and their diameter are in the range of 5?20 ?m,the carboxyl content is about 3.0mmol/g.When the magnetic PHBV microspheres as the carrier were used to detect FN,the detection resuls were also similar with the ELISA plate.At this case,the detection time can be reduced to 3h,because the magnetic separation was used instead of the centrifugal separation.