Multi-functional biosensing chip for rapid determination of herbicides for paddy fields

本研究旨在为今后开发用于评价急性生态毒性指标(荧光或放氧抑制)和慢性生态毒性指标(生长抑制)的集成生物传感芯片奠定基础。1)绿藻固定化方法综述，2)固定化绿藻慢性生态毒性试验，3)绿藻冷冻方法，4)除草剂对绿藻荧光和放氧抑制的定量评价，5)固定化绿藻急性毒性试验，光交联预聚体PVA-SBQ最适合作为绿藻固定化材料。固定化绿藻的生长速度与游离绿藻相似。用固定化绿藻进行长期生态毒性试验，大部分除草剂都能被检测到，用5%二甲基亚砜冷冻的绿藻解冻后具有良好的活力和生长速度。利用这种绿藻也可以进行慢性生态毒性试验。将这一冷冻技术与微板法相结合，可以开发出一种非常简单的新的生态毒性测试方法，光合作用活性的抑制会改变叶绿素荧光强度和放氧速率。用荧光强度变化和氧气浓度变化来评价除草剂的生态毒性。该方法可在1小时内成功测定三嗪类除草剂。最后，在氧电极的敏感区设置绿藻固定膜，并用该电极测定阿特拉津和过氧化氢酶。

This study aims at the basic research for the future development of the integrated biosensing chip for evaluation of acute ecotoxicity indices (inhibition of fluorescence or oxygen evolution) and chronic ecotoxicity index (growth inhibition). In this study, we have investigated the following points : 1)Survey of immobilization method for green algae, 2) Chronic ecotoxicity test by using immobilized green algae, 3) Freezing method of green algae, 4) Quantitative evaluation of inhibitions of fluorescence and oxygen evolution in green algae caused by herbicides, 5) acute toxicity test by using immobilized green algae.As a green algae immobilization material, photo-crosslinkable prepolymer PVA-SbQ was most suitable. Immobilized green algae showed the similar growth rate to free green algae. By chronic ecotoxicity test using immobilized green algae, most of the herbicides could be detected.The green algae frozen with 5% dimethyl sulfoxide showed good viability and growth rate after thawing out. Chronic ecotoxicity test also could be done by using this green algae. By the combination of this freezing technique and micro plate assay method, a quite simple novel ecotoxicity test method could be developed.The inhibition of photosynthetic activity could change chlorophyll-fluorescence intensity and oxygen evolution rate. Ecotoxicity of herbicides was evaluated with fluorescence intensity change and oxygen concentration change. Triazine group herbicides could be successfully determined by this method within an hour.Finally, a green algae immobilized membrane was set at the sensing area of an oxygen electrode, and atrazine and CAT were determined by using this algae-electrode.