Study The Relationship Between The DNA Damage Repair-related Proteins And The Radiation Dose

IntroductionRapid and accurate determination of absorbed dose plays an important role in many fields such as classification diagnosis, clinical treatment of radiation wounded, the precise radiation therapy of cancer patients, biological effect studies induced by radiation and radiation health risk evaluation in special environment. Physical methods of dose evaluation for individual exposure are direct, accurate and authoritative. However, in the situations without wearing personal dosimeter or without installing dose monitoring meters on-site, the physical methods for dose detection are sometimes not possible or time consuming because rebuilding the scene of radiation needs to spend lots time and sometimes the scene of radiation is hard to be rebuild. Thus, in some special or emergency circumstances, to detect personal exposure dose only using physical methods is unrealistic and not practical.Bio-analytical methods to evaluate personnel exposure doses have many advantages and the researches on biodosimeter are the future direction of personal exposed dose survey. However there are still many scientific and technical issues to be resolved in using of biological methods for rapid assessment of personnel exposed dose. First, how to filter out a series of sensitive and specific biomarkers which well response the radiation dose. Second, how to establish a accurate, reliable, highly sensitive and rapid analysis method based on those biomarkers. With the development of new theories, methods and technologies, many new biological indicators in gene and protein levels constantly be discovered and reported. For instance the research shown that content and activity of the ATM protein which was one of DNA damage repair-related proteins had a good dose-effect relationship with radiation dose. ATM plays an important role not only in DNA repairing but also in transporting DNA double-strand break signal. The content of ATM activation is considered to be dependent on the content of DNA double-strand break. When the DNA double-strand break occurs ATM protein itself is phosphorylated at the same time. Meanwhile phosphorylated ATM goes to activate and regulate a series of DNA repair-related proteins, including H2AX, RAD50, NBS1, Mre11, CHk1,2, c-AB1 and makes them gather in the DNA damage site and promotes DNA repair. According to report, the signal of activation of ATM protein can be seen at 30 minutes after 0.25Gy irradiation. Further more a high brilliant fluorescence foci of activation of ATM protein can still be detected at 3 hours after 4Gy irradiation. In the past, people usually used the immunofluorescence method to detect the ATM activation. Although this method is relatively straightforward in observation of ATM activation in cells it can only do semi-quantitative detection. Therefore it is difficult to establish accurate relationship between amount of protein activation and dose-effect, especially in the situations of low irradiation dose range and innitial radiation stage. Accordingly, more sensitive and specific detection methods need in response to further study which is the relationship between ATM protein expression or activation and radiation dose. Currently nano-materials have been widely used in the areas of DNA and protein sensors because of their excellent biocompatibility and unique optical properties. In this study, we used a unique designed nano-material as probe which is modified by the chemical or biological to realize sensitive and specific detection to ATM protein...The basic steps of the study are as follows:first, the aldehyde bead surface is modified by ATM monoclonal antibody and ATM multi-antibody to capture the antigen in the samples by forming a monoclonal antibody-antigen-multi-antibody sandwich structure. And then nano-bio-probe is added into reaction system to achieve signal amplification and output. In this study three nano-bio-probes such as IgG-gold nanoparticle-DNA probe, IgG-nano-Au-HRP probe and IgG-carbon nanotubes-HRP probe are involved. Among them a simple, stable and reliable method also is selected to achieve the objective.Contents:Sensitive and reliable methods of ATM protein detecting are studied based on modified nano-materials as probes combined with magnetic bead separation and enzyme-linked immunosorbent technology. According to the different probe preparations the research is divided into three parts:1. The research of IgG-Nano-Au-HRP probe for ATM protein detection2. The research of method based on IgG-carbon nanotubes-HRP probe.3. The research of RCA method based on the IgG-gold nanoparticle-DNA probe. Methods and Measures:1. Making of gold nano particles and carbon nanotubes probeAdjust the pH of the gold nanoparticles solution near the isoelectric point of adding protein and activate the carboxyl of carbon nanotube using EDC and NHS respectely. Then IgG at 0.005 mg/ml and HRP at 1 mg/ml was added to prepare gold nanoparticles or nano-carbon tube mixture and stirred overnight at room temperature. The probe was blocked with BSA and washed to remove any free HRP and IgG and the preparations were stored in refrigerator at 4℃by 0.5%casein for use.2. Check of prepared probeTo check if proteins which banded on the surface of the probe possess activity, ATM polyclonal antibodies-coated magnetic beads (blocked with 0.5% casein,0.1M PBS, PH7.4) were coated with a solution of the probe. The successful connection of the two proteins and biological activity of the probe were detected by the specific color reaction between IgG connected with HRP on the probe and polyclonal antibody of ATM on the magnetic beads when TMB was added into system.3. Detection of target protein by the probeATM monoclonal antibodies-coated magnetic beads (blocked with 0.5% casein,0.1 M PBS, pH 7.4) were coated with a solution of ATM antigen in PBS at 37℃for 1 h, washed four times in PBS/0.25% Tween20. ATM-polyclonal antibodies were than added into, at 37℃for 1 h. After incubating and washing, probe was added in, and incubated at 37℃for 1h. The HRP on the probe was detected by TMB.4. Data analysisSoftware SPSS11.0 was Used to analyse the data. One-dimensional analysis of variance for significant test (as P<0.05 for significant difference). The charts produced using origi8.0 software. The threshold of detecting limitation was identified as the mean of the negative control plus three times standard deviation of the negative control. The signal is higher than the concentration of this value shall be the minimum detection limit of the method. Method-specific can be obtained from the difference between the experimental group and the embodiment.Results SWNT immunosensors can accurately detect the ATM in standered samples with very high sensitivity and selectivity. Further, the immunosensors have a very good reproducibility, as demonstrated by device-to-device standard deviations with both Ab2-CNT-HRP bioconjugates and conventional single-label HRP-Ab2. The best sensitivity is obtained using the Ab2-CNT-HRP bioconjugates with high enzyme label/Ab2 ratios for signal amplification.Preliminary ConclusionsPresent methods for the detection of trace protein have their own new ideas and strengths. In this research application of carbon nanotube probe in trace protein detecting shows a certain stability and character of signal amplification compared with other methods. This method guaranteed not increasing the experimental procedures like RCA method based on the IgG-gold nanoparticle-DNA probe, IgG-Nano-Au-HRP probe and the classical ELISA method did and keeping the advantage inheriting from the electrochemical system. Using this kind of probe tactics we successfully achieved the specific and sensitive detection of ATM in an optimizing system which detection limit was lower 1 pg/ml.