The Setup And Application Of New Solid-phase NF-κB Detection System

NF-κB, a collective name of dimeric transcription factors, is composed of members of the Rel family proteins that recognize and bind a specific DNA sequence. NF-κB is involved in a lot of physiological processes such as immunity, inflammation, cell proliferation, apoptosis and even tumorigenesis by regulating the transcription of a larger number of genes. Targets of NF-κB and regulating signal pathway have used for new drug development and disease diagnosis. However, the traditional methods, such as EMSA, can not meet the fast and high throughput analysis need in modern life science and medicine. The development of new typical detection NF-κB of would greatly improve the research process of modern life science and medicine. The development of modern biotechnique and biomolecule immobilization technique provide a powerful support for detection of the specific biomolecule. The double-stranded DNA-couplate (dsDNA-coupled) microplate had been manufactured by covalently immobilizing an amino-modified oligonucleotide to the wells of plate coated with N-oxysuccinimide esters, and then annealing a complementary oligonucleotide. The optimization of parameters had been studied. The immobilization efficiency and NF-κB detection of the two different immobilization ways of DNA probes had been compared with EMSA. The ECV304 cells were cultured with natural antioxydants and drugs, and then TNF-αinduced. The cells extracts were detected with the dsDNA-coupled microplate and chemiluminescent EMSA. The results indicated that the detections of dsDNA-coupled microplate were in agreement with that of the chemiluminescent EMSA. This homemade dsDNA-coupled microplate would provide a simple and versatile platform for studying DNA-binding proteins.Recently, nanoparticles self-assembly technique has been applied in the biological detection and received great attention because of the good control of nanoparticles self-assembly. In this paper. PMMA nanoparticles had been coated onto the glass substrate by the lifting self-assembly. Then the interspaces of opal film were filled with titania nanoparticles. After calcination, the TiO₂ inversed opal film biosensor had been structured .By the optical interference spectroscopy. Scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS) had been demonstrated the fabrication of the TiO₂ inverse opal films. And then the TiO₂ inverse opal films had been used in organic solvents, immunological assay and NF-κB detection. This method provided a new way for structuring free-label biosensor.The mayor contributions were described as followed:1. The setup of dsDNA-coupled microplate for detection NF-κB systemThe double-stranded DNA-couplate microplate had been manufactured by covalently immobilizing an amino-modified oligonucleotide to the wells of plate coated with N-oxysuccinimide esters, and then annealing a complementary oligonucleotide. The coupled DNA probes concentration would affect the signal of NF-κB detection. The signal of 10ng NF-κB detection could reach to the maximum at 75pmol/100μL-well DNA probes concentration. This NF-κB assay system had good linear detection relationship in 4-70 ng/50μL-wel 1 NF-κB concentration range (R²ï¼ž0.99). Not only the purified N F-κB but also the cells extracts had been quantified. The linear detection concentration was 0.6251ag TNF-αinduced cells extraction, 1.25μg un-induced cells extraction, and 4ng the purified NF-κB. The specificity was researched with the purified NF-κB and cells extraction. The results showed the dsDNA coupled microplate had good specificity for NF-κB and could be used to assay for different binding sites for the DNA binding protein. This dsDNA coupled microplate had a good repetition and could be applied to through-put detection for NF-κB.2. The optimization of dsDNA coupled microplate system for detection of NF-κBThe assay conditions of such dsDNA-coupled plate were fully optimized for detecting a very important transcription factor NF-κB. The optimized parameters of dcPlate for assay of NF-κB were as follows: Immobilized DNA probe at the concentration of 25 pmol/well, incubation time of 90 min for NF-κB binding to dcPlate, primary and secondary antibody concentration of 1:1000 dilution, incubation time of 90 min for primary antibody binding to NF-κB, temperature of 25℃for the whole process. Under the optimized conditions, the signal was improved three times higher than that from not optimized conditions. The linear detection range was from 4.69 to 75 ng/well for half hour colorimetric development. The non-specific DNA probes were not needed in the procedure of assay for cell extraction because of the interference of other protein.3. The three label methods for detection NF-κB have been compared and applied in the screen of medicineThe comparison of N-oxysuccinimide esters coupled microplate with streptavidin microplates, the immobilization of DNA probes was less in the former. But, the former was more stable and cheaper than the latter. The detection linear range of the former was narrower than that of the latter. The detection linear concentrations of two assay systems were lower than that of chemiluminescent EMSA. The results of two microplate assay systems for NF-κBp50 were consistent in the two natural antioxydants-curcumin and nordihydroguaiaretic acid direct inhibition DNA binding by NF-κB in vitro and were in agreement with previous reports. Two new drugs-ruscogenin and diosgenin were used to inhibit DNA binding by NF-κB in vivo. The results showed that both of them could clearly inhibit NF-κB transference by N-oxysuccinimide esters coupled microplate assay system and chemiluminescent EMSA system. The results of two assays systems were also consistent in the dexamethasone. These indicated this assay system could be used in screen of NF-κB-related medicine.The method for analysis of NF-kappaB DNA binding activity has advantages over the EMSA: (1) Higher speed: 3-5h post sample preparation, (2) Simplicity (3) Greater sensitivity: 4 NF-kappaB/well, (4) Quantitative: linear range: 4-70ng NF-kappaB; R²= 0.997 (5) High throughput adaptability: 96-well plate format can analyze up to 40 samples in duplicate, (6) Safety: No radioactive isotopes.4. nanoparticles self-assembly and setup of the new typical free-label biosensorThree different nanoparticles were self-assemblied onto the glass surface by the lifting method to pack to be different opal films. The parameters of self-assembly were optimized. The principle of different self-assembly packing styles was discussed. The 3DOM TiO₂ inversed opal film sensor was fabricated. The results demonstrated that soft PMMA nanoparticles could pack to be coexist of FCC (111) plane and FCC (100) plane. After optimization of self-assembly parameters, the area of FCC (100) plane could reach to 200μm×200μm. The packed styles were closely related with nanoparticles materials. The self-assembly principle of soft PMMA nanopartcles accorded with minimum- touch-area principle and also the reason of maximum entropy. The TiO₂ inversed opal film was fabricated by the infiltration of the titania nanoparticles in the close-packed FCC(111)opal film structure and calcination at 500℃for 1h. SEM images, reflected spectrum, and XPS showed that the TiO₂ inversed opal film with ordered structure was fabricated by this process. 5. Application of 3DOM TiO₂ inversed opal film sensor3DOM TiO₂ inversed opal film was used as the sensoring substrate of refractive index sensor. The sensor could be used to quantify and quality in the different refractive index organic solvents. Compared with the PS inversed opal film and SiO₂ inversed opal film sensors, the 3DOM YiO₂ inversed opal film sensor has the higher sensitivity and could be reused. The resolution of the sensor could reach to 0.007 refractive index. The 3DOM TiO₂ inversed opal film sensor has been applied in the free-label biosensor. The experimental results showed that the sensitivity of 3DOM TiO₂ inverse opal film can be reached to 460nm/refractive index unit (RIU) and 0.6μM (equivalent to 0.15ngmm^-2) protein concentration detection limit. The detection linear range was from 150 pgmm^-2 to 3.0ng mm^-2. After the 3DOM TiO₂ inverse opal film was modified with aminosilane and glutaraldehyde, the double-stranded DNA was immobilized on the surface of the microcavity. The detection limit of NF-κB was 20 nM with good specificity. The assay system can be used as primary platform for studying the DNA binding protein.