It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and
husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research
combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method,
to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for
detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection.
Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of
hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two
Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane
mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show
that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and
lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.
An electrochemiluminescence non-PCR method has been developed for the detection of genetically modified
organisms (GMOs) in crops. Genomic DNA of GMOs was digested with two restriction endonucleases (FOK I and
BsrD I), and hybridized with three Ru(bpy)32+ (TBR)-labeled and one biotinylated probes. The hybridization products
were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence
(ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL
signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol,
and the GM components can be clearly identified in GM tobaccos. The ECL non-PCR method will provide a new means
in GMOs detection due to its safety, simplicity and high efficiency.
Simpler, cheaper method for DNA sequence recognition is of a greater scientific and economic interest. In this paper,
we develop a novel colorimetric detection method for recognition of specific DNA. The detection method bases on the
strong distance-dependent optical properties of gold nanoparticles (Au-nps) and the DNA sequences' electrostatic
interactions with the Au-nps. As a result of the single-stranded DNA (ssDNA) displaying far higher affinities than
double-stranded DNA (dsDNA) to negatively charged Au-nps, when the concentration of salt is added, Au-nps in
ssDNA keep stable because of the absorption of large amount of ssDNA, the mixture's color is red. But Au-nps in
dsDNA aggregate immediately, and the mixture's color turns out to be blue. Therefore, we can judge whether the
hybridization occurs by the mixture's color or absorption spectrums. Au-nps aggregation in this detection mode is not
induced by hybridization of target DNA which cross-links the nanoparticles but by salt, then the color transition is rapid
and clearly visible. This method is simple, cheap, and we can detect about 2Opmol specific DNA sequences.
Reactive oxygen species (ROS) is thought to play an important role in cell signaling of apoptosis, necrosis, and
proliferation. Light irradiation increases mitochondrial reactive oxygen species (ROS) production and mediates its
intracellular signaling by adjusting the redox potential in tumor cells. Mitochondria are the main source of ROS in the
living cell. Superoxide anions (02- are likely the first ROS generated in the mitochondria following radiation damage,
and then convert to hydrogen peroxide (H202), hydroxyl radical (•OH), and singlet oxygen (102), etc. Conventional
methods for research ROS production in mitochondria mostly use isolated mitochondria rather than mitochondria in
living cells. In this study, a highly selective probe to detect mitochondrial 02- in live cells, MitoSOXTM Red, was applied
to quantify the mitochondrial ROS production in human lung adenocarcinoma cells (ASTC-a-1) with laser scanning
microscope (LSM) after ultraviolet C (UVC) and He-Ne laser irradiation. Dichiorodihydrofluoresein diacetate (DCFHDA),
a common used fluorescent probe for ROS detection without specificity, were used as a comparison to image the
ROS production. The fluorescent image of MItoSOXTM Red counterstained with MitoTracker Deep Red 633, a
mitochondria selective probe, shows that the mitochondrial ROS production increases distinctly after UVC and He-Ne
laser irradiation. DCFH-DA diffuses labeling throughout the cell though its fluorescence increases markedly too. In
conclusion, the fluorescent method with MitoSOXTM Red reagent is proved to be a promising technique to research the
role of ROS in radiation induced apoptosis.
A new method for identification of point mutations was proposed. Polymerase chain reaction (PCR) amplification of a sequence from genomic DNA was followed by digestion with a kind of restriction enzyme, which only cut the wild-type amplicon containing its recognition site. Reaction products were detected by electrochemiluminescence (ECL) assay after adsorption of the resulting DNA duplexes to the solid phase. One strand of PCR products carries biotin to be bound on a streptavidin-coated microbead for sample selection. Another strand carries Ru(bpy)32+ (TBR) to react with tripropylamine (TPA) to emit light for ECL detection. The method was applied to detect a specific point mutation in H-ras oncogene in T24 cell line. The results show that the detection limit for H-ras amplicon is 100 fmol and the linear range is more than 3 orders of magnitude, thus, make quantitative analysis possible. The genotype can be clearly discriminated. Results of the study suggest that ECL-PCR is a feasible quantitative method for safe, sensitive and rapid detection of point mutation in human genes.
Oxidative stress is mainly mediated by reactive oxygen species (ROS). Evaluation of oxidative stress is helpful for choosing an appropriate method to protect the organism from the oxidative damage. In this study, a highly sensitive and simple chemiluminescence method is presented for the evaluation of radiation-induced oxidative stress in human peripheral lymphocytes. The lymphocytes were irradiated by ultraviolet radiation (320-400nm, UVA) with different doses. The ROS generated by the lymphocytes was detected by chemiluminescence method, using a highly sensitive chemiluminescence probe 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-α] pyrazin-3-one (MCLA). The cell viability was detected with Cell Counting Kit-8 (CCK-8). The malondialdehyde (MDA), a marker of lipid peroxidation and oxidative stress, and the total antioxidant capacity (TAC), a parameter that is taken as evidence of oxidative stress, were measured too. The results show that both chemiluminescence intensity, cell mortality and MDA concentration of lymphocytes grow with the increase of UVA dose range from 0.5 to 8 J/cm2, while the TAC decreases. There exists a positive relationship between cell oxidative damage degree and the chemiluminescence intensity of lymphocytes. This highly sensitive chemiluminescence method would potentially provide an easy way to evaluate the level of UVA-induced oxidative stress readily, sensitively and rapidly
The detection methods for genetically modified (GM) components in foods have been developed recently. But many of them are complicated and time-consuming; some of them need to use the carcinogenic substance, and can’t avoid false-positive results. In this study, an electrochemiluminescence polymerase chain reaction (ECL-PCR) method for detection GM tobaccos is proposed. The Cauliflower mosaic virus 35S (CaMV35S) promoter was amplified by PCR, Then hybridized with a Ru(bpy)32+ (TBR)-labeled and a biotinylated probe. The hybridization products were captured onto streptavidin-coated paramagnetic beads, and detected by measuring the electrochemiluminescence (ECL) signal of the TBR label. Whether the tobaccos contain GM components was discriminated by detecting the ECL signal of CaMV35S promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM tobaccos. The ECL-PCR method provide a new means in GMOs detection due to its safety, simplicity and high efficiency.
Chemiluminescence (CL) is a highly sensitive detection method with broad biological applications. In this work, a CL detection system is developed and applied in detection the CL from human lymphocyte induced with Co-60 irradiation, for the first time. The radiation damage of lymphocyte was detected by cell counting and MTT method for various irradiation doses. The results show a good correlation between the CL intensity and the radiation-induced damage of lymphocyte. Cell activity increased gradually with the increase of radiate dose, when the dose was under 3Gy. When the radiate dose was above 3Gy, the results were contrary. The cell counting results corresponded well with the MTT method. The CL detection method, thus, may provide an alternative way in evaluation of radiation damage.
In this work, we have designed an electrochemiluminescence (ECL) reaction system used to indicate P53 protein from human lung cancer cells. And we optimized the working electrode firstly in this experiment. The electrochemiluminescence magnetic immunoassay is quantitative, reproducible, and requires only minimal sample processing. A single photon counter was used to heighten the ECL detection limit. The homologous immunoassay format consisted of a double antibody sandwich in which a biotinylated capture antibody, pre-bound to streptavidin-coated magnetic beads, was used to bind antigen from test samples. A detector antibody, labeled with ruthenium( II ) tris-bipyridal chelate, was added and, when bounded to the bead immunocomplex, light was generated in the presence of an excess of tripropylamine. The light was detected and measured by the ECL reaction system we designed. High-sensitive, rapid and reproducible detection of tumor marker P53 protein was actualize with this detection system. The results showed that the electrode with many holes on it can enhance the ECL efficiency, so the detection limit of the system can be improved to 1/1000 fmol/L free labeled Ru(bpy)32+. Ru(bpy)32 + TPA(tripropylmine) ECL reaction system was used to indicate P53 protein from human lung cancer cells. This method could have potential applications in early-stage tumor diagnosis and environmental inspection.