Luminescent Bacteria Toxicity Assay Based On Microfluidic Devices

The rapid development of China's industrial strength also brings a series of environmental problems.A series of pollutants produced in the process of industrial production enter into the surrounding environment with waste gas,waste water and waste residue,which poses a potential threat to the health and safety of people.The detection of acute toxicit y of water using luminescent bacteria can directly and comprehensively reflect the toxic effects of pollutants on human body.The luminescent bacteria method is not only simple and fast,but also easy to operate.It is easy to be coupled with many optical elements and has good reproducibility.However,the existing equipment is complex and time-consuming,and is not suitable for on-line continuous monitoring.In addition,the luminescent intensity of the luminescent bacteria after the recovery is unstable,which makes the detection result more error.The aim of this study is to establish a new type of acute toxicit y analysis and detection device based on microfluidic technology with Vibrio qinghaiensis sp.Q67,and to promote the development of the on-line continuous detection direction in the area of the acute toxicity detection method based on luminescent bacteria.In this paper,a water quality acute toxicity detection device based on 2 ml centrifuge tube was established,it was used as as a contrast detection system.Through the improvement of sample room design,light detection system and sampling system,the device can run stably and efficiently.The device was used to determine the luminescence intensity of different OD600 values,so as to determine the resolution of the light signal,and the relative standard deviation of the results obtained by 3 repeated experiments was 2.1%.In addition,the basic detection conditions for the best luminescence of Vibrio qinghaiensis sp.Q67 were determined,that is,using the physiological saline as the suspension,regulating the p H of the water sample to be 7.0~9.0,and controlling the dosage of the bacterial liquid at 200 ?l.The device was used to detect the acute toxicity of different concentrations of Zn2+? Cd2+ on the water quality of Vibrio qinghaiensis sp.Q67,and the semi lethal concentration EC50 of Zn2+ and Cd2+ was obtained.A microfluidic chip with toxicity analysis is designed and optimized.The micro mixer is optimized and the mixing performance is improved.The entrance angle is 90 °,the width of the channel is 100 ?m,and the chip thickness is 100 ?m.The COMSOL software is used to simulate the concentration of the two fluid components on the cross section of the channel X-Y,and the micro mixer structure with better mixing performance is determined.The difference between the mixing performance of different length straight channels and two different structural curves is compared,and the corner curve is chosen as the hybrid structure of the chip,and the number of the mixed structure is determined.The two fluid components are fully mixed into the chip detection area,the detection area is hexagonal structure,the length is about 10 mm,and the width is about 10 mm.After the detection,the microchip is flushed with saline.Finally,a new water quality acute toxicity analysis and detection device based on microfluidic chip was established.The acute toxicity of several typical pollutants to Vibrio Qinghai was tested.The resolution of the new device for different OD600 values and the reproducibilit y of the three test results(the relative standard deviation of 0.7%)were verified.Subsequently,an acute toxicity test method for 5 kinds of heavy metals,3 antibiotics and 3 dyes was established,and the dose effect curves of these 11 pollutants on Vibrio qinghaiensis sp.Q67 were obtained.The EC50 of each pollutant in 5 min,10 min and 15 min respectively was obtained.The rules of the toxicity of these pollutants are as follows: Cu2+>Pb2+>Cd2+>Zn2+>Ni2+,ciprofloxacin > norfloxacin > penicillin G,Luo Danming B> orange G> methylene blue.The different mechanisms of toxicity of three substances such as heavy metals,antibiotics and dyes to Vibrio qinghaiensis sp.Q67 are clearly defined,heavy metals inhibit the luminescence of Vibrio qinghaiensis sp.Q67 by inhibiting the synthesis of luciferase,antibiotics inhibit the luminescence of Vibrio qinghaiensis sp.Q67 by destroying cell structure,causing bacterial death or inhibiting bacterial activity,the toxicity of dye molecules is mainly related to the type of dyes,the size of molecules and the properties of functional groups.It is proved that the test results of the new device are more stable in time and the relative standard deviation of the test results is lower.