Development of Bioelectrical Microsensor for Waterborne Radioactivity Detection

Abstract

Ionizing radiation can cause biochemical changes in cells and consequently it will lead to sickness, cancer or even death, by breaking the DNA structure directly or inducing oxidative free radicals to damage DNA molecules indirectly. Waterborne radioactive contaminants in water or food pose a serious global threat. The objective of this study is to develop a microfluidics platform for rapid and accurate detection of radioactivity in waterborne drinking water and food sample.

D. radiodurans is a gram-positive bacterium, which shows extreme resistance to ionizing radiation due to its remarkable DNA repair system. It can survive cold, dehydration, acid, and saline, which makes it an ideal model for radiotoxicological study. The response of various genes against the gamma radiation, alpha radiation and sodium selenite-induced oxidative stress was studied by real-time PCR. These genes are either responsible for the DNA repair, DNA protection or anti-oxidative stress. Based on the real-time PCR result and the recent transcriptome analysis, DNA damage responsive gene A (dr_0423, DdrA), which encoded for a protein to protect the DNA from nucleolytic degradation after double strand break, showed a sensitive response with a 5-fold expression upregulation against gamma radiation of 400 Gy. It also showed a dose-dependent upregulation against both gamma radiation, alpha radiation and sodium selenite -induced oxidative stress. This result highlighted the potential of using dr_0423 as a sensitive biomarker for transgenic study and radiation detection.

Thus, dr_0423 genomic DNA sequence including ca. 6000 bp 5’upstream region was cloned, and several putative cis-regulatory elements were identified by in silico study. A transgenic strain carrying the construct harboring the 6000 bp 5’upstream region and a GFP reporter gene was generated. However, the basal GFP signal was very high and it failed to show any induction difference of GFP signal against the oxidative stress. Another transgenic strain, designated DRPZ423, was generated which carry the construct harboring the first 300 bp 5’ upstream region containing two Radiation Desiccation Response Motif-Like (RDRM) sequences and a beta-galactosidase reporter gene. Beta-galactosidase activity assay demonstrated that the strain DRPZ423 was able to express beta-galactosidase in response to the oxidative stress induced by sodium selenite and gamma radiation in a dose-dependent manner. However, the detection limit of this biosensor was not good enough to detect low dose radiation as it failed to detect the gamma radiation of 2 Gy. Further improvement was made in this study to enhance the sensitivity and detection limit. Microfluidics system was therefore, employed to improve the sensitivity of the biosensor DRPZ423. It also enabled miniaturization, whereas the reaction could be carried out inside the chip. In our study, the platform allowed cell-trapping, sensor-sample compartmentalization and amperometric detection and it is operated with three phases: bacteria trapping (phase I), radiation loading and acting on bacteria (phase II) and electrochemical analysis (phase III). For phase I, the bacteria suspension was loaded into the chip and concentrated by the micro-concentrator, which composed of 4 electrodes. By the applied voltage of 20 Vp-p with a frequency of 300 kHz, the dielectrophoretic force would be generated to concentrate the bacteria into the irradiation region. Thus, the number of irradiated cells was increased among the population, which help to enhance the sensitivity. For phase II, the testing sample was anchored inversely on the chip and irradiate the trapped bacteria without mixing through the ultra-thin transparent membrane (thickness: 2 µm) on the bottom of the chamber. This ultra-thin membrane was used to insulate the bacteria biosensor from the measuring sample, which facilitate the elimination of the false target signal, as the transgenic strain DRPZ423 could also show signal induction against other contaminants in the measuring sample that poses genotoxicity. For phase III, PAPG was loaded into the chip and mixed with the irradiated bacteria. The PAPG would react with the beta-galactosidase induced by the irradiation within the cell and converted into PAP. The PAP would subsequently diffuse out of the cell and oxidized into para-quinonimine at the electrode surface. The oxidative current was measured by the electrodes of the detection chamber by amperometric detection as the bioresponse of DRPZ423 to the radiation.

The effectiveness of measuring ionizing radiation using this detection platform was investigated. Significant dose-dependent induction of current signal was observed against various doses of gamma irradiation (0 Gy, 2 Gy, 10 Gy and 20 Gy), indicated that the sensitivity was successfully improved by employing the amperometric detection using microfluidics technology. Due to the unavailability of waterborne alpha radiation sample, the effectiveness of alpha radiation detection was performed without the cell concentrating process. Result showed showed a similar induction against the alpha irradiation with different duration (0 s, 10 s, 60 s and 360 s). However, it failed to discriminate the 10 seconds alpha irradiation.

To conclude, this study successfully developed the first microfluidics system coupling with microbial biosensor for ionizing radiation detection. The system showed quick response to a wide range of ionizing radiation with different penetrating power in the sample promptly and directly. All my findings indicated the great practical potential of this platform as a simple, rapid, and economic method for waterborne radioactivity in drinking water or food sample.

Date of Award	29 Jan 2018
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Hiu Wai Raymond LAM (Supervisor) & Shuk Han CHENG (Co-supervisor)