The Research Of Detection For PML/RARa Fusion Gene With Functionalized Graphene Oxide

ObjectiveGraphene is the world’s thinnest novel two-dimensional nanomaterials with a thickness of only0.35nm. It becomes a hotspot in the field of nanotechnology and has many important potential applications in the field of composite materials, sensors and energy, etc, owing to its excellent electrical, mechanical and thermal properties. Geim and Novoselov, the discoverers of graphene, won the2010Nobel Prize in Physics. The research of graphene in the biomedical application began in the recent years. The graphene oxide (GO), an oxidative derivative of graphene, has oxygen-containing functional groups in graphene edge or plane, such as hydroxyl, carbonyl, carboxyl. The presence of these functional groups in GO makes it biocompatible, stable in hydrophilic solution, and compatible with polymers. These groups are also conducive to the chemical functionalization to achieve targeted applications in different areas, especially the biomedical field.At present, many scholars have studied on the application of the GO in drug loading systems, biological detection, bio-imaging and cancer treatment as well as its biological safety. In the biological detection, the GO can be used to detect DNA, small molecules and proteins, etc. GO is a carrier and has the characteristics of quenching fluorophores in the detection.Acute promyelocytic leukemia (APL) is an acute leukemia, which is often accompanied by severe bleeding. More than95%APL results from the specific chromosomal translocation t (15;17)(q22; q12～21) so that the promyelocytic leukemia (PML) gene on the15th chromosome and the retinoic acid receptor a (RARa) gene on the17th chromosome fuse together to form the PML/RARa fusion gene, which is a marker of APL in the early diagnosis and prognostic monitoring. The detection methods of the PML/RARa fusion gene include chromosome analysis, real-time quantitative RT-PCR, FISH, etc. But low specificity, complicated operation and the high cost of these testing methods limit their wide applications.GO has a high adsorption capacity for the probe with fluoresce-labeled single-stranded DNA(ssDNA), and can quench its fluorescence. In the presence of a target molecule, the ssDNA labeled by the fluorescent, can be detached from the GO, and the fluorescence of the probe is restored. So GO can be used to the detection of the PML/RARa fusion gene by measuring fluorescence intensity.Methods1. The preparation of functionalized GO1.1Prepare GO which small size100nm was Suitable for using in biology field.1.2The functionalization of GO:GO Was modified by NH2-PEG-NH2.2. Fluorescence spectrophotometer to detect fluorescenceDesign probe according to alternative splicing isoform E5(-)E6(-) of PML/RARa fusion gene. Because alternative splicing isoform E5(-)E6(-) has the longest cycle after ATRA induced. It is the most sensitive in monitoring dynamic change of fusion gene. Prepare fluorescent probe with green fluorescent protein(GFP) mark ssDNA.Prepare GO owns different concentrations in order to^observe quenching function and the fluorescence recovery when target molecule appears.3. Cell cultureThe experimental NB4group cells and the control K562group cells were seeded in RPMI-1640culture medium containing10%fetal bovine serum and cultured at37℃with5%CO2with half of the culture medium exchanged after28～3days. Before the experimental and control cells were counted the density of the cells was adjusted according to the experimental requirements.4. Functionalized GO load probes into living cells.NB4cells and K562cells incubate with GO、fluorescent probes and buffer mixture. Laser confocal microscopy and flow cytometry instrument to detect.5. Functionalized GO load fluorescent probe into the cell which drilled hole. Fixed NB4cells and K562cells With Punch reagent I firstly, then honeylocust the cell membrane with punch reagent Ⅱ. Incubation fluids (divided into intervention group and no intervention group of fluorescent probe) incubate with NB4cells and K562cells. Finally using Laser confocal fluorescence microscopy and flow cytometry instrument to detect.6. Application research of functional GO in the diagnosis of APLCollect of36cases inpatient and outpatient cases of the Second Hospital of Shanxi Medical University. Among them,16cases which PML-RARa positive;20cases which PML-RARa negative. Join in CD45, fixed fluid, drilling fluid and centrifugal. Mixed with GO, fluorescent probes, buffer liquid wait for1h.At last, samples was cleanied by PBS (1x) buffer, centrifuged, detected GFP by cytometry instrument.Results1. The preparation of functionalized GOPrepared GO for5days. GO color changed from blackish green color to the the bright yellow, and last was brown, testing samples was tested by SPA300HV scanning probe microscope. GO size close to100nm. GO Was modified by NH2-PEG-NH2, and then use FTIR-8400s Fourier infrared spectrum characterized. Biocompatibility experiments showed that the functional can GO will be stable in PBS solution cell culture medium、serum and GO wiil not precipitate after10000r high-speed centrifugal.2. Fluorescence spectrophotometer to detect fluorescenceDifferent concentration of GO has different quenching ability for fluorescence. In our experiment, the concentration of0.04mg/ml is the best. In this concentration, target molecules concentration was arranged from high to low (300nm,100nm,50nm) correspondingly, fluorescence intensity was from strong to the weak when target molecules occurs. GO meets the target molecule fluorescence will not recover if the concentration is greater than0.1mg/ml. 3.Functionalized GO load probes into cells:Test results with Laser confocal microscopy and flow cytometry instrument.3.1In our experiment, GO did not ake fluorescent into living cells.3.2The experiment for drilled cells:NB4cells was experimental gruop; K562cells was control group. Only NB4cells have fluorescent signal When fluorescent probe intervention,while others have no this phenomenon. Linear probe and the hairpin probe have no obvious difference. The hairpin probe is more stable, so we select hairpin probe. Probes concentration respectively choose150nM,200nM,250nM. In three kinds of concentration, the concentration200nM was more ideal. Incubation fluid and cell incubate for1h can be tested. The fluorescent will not quench after sample preparation, the test results will not be affected. Using this method can find the number of positive cells account for more than58.6%(P<0.0001).4. Application research of functional GO in the diagnosis of APLUsing functional GO carry negative fluorescent probe to detect whether bone marrow samples contain L type PML/RARa fusion gene. Under the intervention of probe, only cells which contain L type PML/RARa fusion gene will appear fluorescent signals, Whereas no signals (P<0.0001). Premyelocyte、red blood cell、 lymphocyte was divided3groups by Flow cytometry instrument. Find fluorescence intensity compared between groups, the experimental results show that there were no difference between3groups of not containing L type PML/RARa fusion gene (P>0.05), but there were significant differences in L type PML/RARa fusion gene positive group(P<0.0001).Comparing between Groups, the fluorescence intensity of red blood cell and lymphocyte has no difference(P>0.05), the fluorescence intensity of Premyelocyte is higher than red blood cell and lymphocyte (P<0.0001). When we don’t know if the bone marrow specimens contain PML/RARa fusion gene or not, whether fluorescence intensity or fluorescence positive cells number all have values in clinical diagnosis (P<0.05), Diagnostic accuracy was92.2%,90.9%respectively.There are differences between GO and PCR detection method (P<0.05). Conclusion1. Nano GO functioned by double amino polyethylene glycol has good biological compatibility, and will not togethe in biological solution.2. Extracellular experiments shows that GO loaded ssDNA fluorescent probes could identify the target molecule accurately.3. L type PML/RARa fusion gene of NB4cell lines can be specific identified by functional GO.4. In the study of clinical APL diagnosis, functional GO as the carrier, loaded with fluorescent probe can identificate PML/RARa fusion gene conveniently and quickly.This method is cheap、no pollution. According this theory, we can design different probe and use the GO to test. Provide a new thought for clinical to develop rapid and accurate diagnostic method.