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

本研究旨在为开发用于评价急性生态毒性指标（荧光或氧释放抑制）和慢性生态毒性指标（生长抑制）的集成生物传感芯片进行基础研究。在这项研究中，我们调查了以下几点：1）绿色的固定化方法综述，2）固定化绿色的慢性生态毒性试验，3）绿色的冷冻方法，4）除草剂对绿色荧光和放氧抑制的定量评价，5）固定化绿色的急性毒性试验。光交联预聚物PVA-SbQ是最合适的。固定化绿色藻的生长速率与游离绿色藻的生长速率相近。用固定化绿色藻类进行的慢性毒性试验表明，大多数除草剂都能被检出，用5%二甲基亚砜冷冻的绿色藻类解冻后具有良好的存活力和生长速度。利用该绿色藻类也可进行慢性生态毒性试验。将此冷冻技术与微板法相结合，可建立一种简便的新的生态毒性检测方法：光合活性的抑制可改变叶绿素荧光强度和放氧速率。用荧光强度变化和氧浓度变化评价除草剂的生态毒性。最后，将绿色藻类固定化膜置于氧电极的敏感区，利用该电极对莠去津和过氧化氢酶进行了测定。

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.