Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B
Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was...
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| Format: | Article |
| Language: | English |
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MDPI AG
2018-12-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/8/4/126 |
| _version_ | 1819289604322230272 |
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| author | Zhaohui Wang Pak Kin Wong |
| author_facet | Zhaohui Wang Pak Kin Wong |
| author_sort | Zhaohui Wang |
| collection | DOAJ |
| description | Nuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA⁻protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems. |
| first_indexed | 2024-12-24T03:09:29Z |
| format | Article |
| id | doaj.art-1ee9cf30cf5243a2bf9d8cad0556c6e0 |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-12-24T03:09:29Z |
| publishDate | 2018-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-1ee9cf30cf5243a2bf9d8cad0556c6e02022-12-21T17:17:51ZengMDPI AGBiosensors2079-63742018-12-018412610.3390/bios8040126bios8040126Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa BZhaohui Wang0Pak Kin Wong1Department of Electrical Engineering and Computer Science, Texas A&M University-Kingsville, Kingsville, TX 78363, USADepartment of Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USANuclear factor kappa B (NF-κB), regulating the expression of several genes that mediate the inflammatory responses and cell proliferation, is one of the therapeutic targets for chronic inflammatory disease and cancer. A novel molecular binding scheme for the detection of NF-κB was investigated for its affinity to Ig-κB DNA composed by dye and quencher fluorophores, and this specificity is confirmed by competing with the DNA sequence that is complementary to the Ig-κB DNA. We create a normalization equation to remove the negative effects from the various initial fluorophore concentrations and the background noise. We also found that a periodic shaking at a frequency could help to stabilize the DNA⁻protein binding. The calibration experiment, using purified p50 (NF-κB), shows that this molecular probe biosensor has a detection limit on the order of nanomolar. The limit of detection is determined by the binding performance of dye and quencher oligonucleotides, and only a small portion of probes are stabilized by DNA-binding protein NF-κB. The specificity experiment also shows that p50/p65 heterodimer has the highest affinity for Ig-κB DNA; p65 homodimer binds with intermediate affinity, whereas p50 shows the lowest binding affinity, and Ig-κB DNA is not sensitive to BSA (bovine albumin serum). The experiment of HeLa nuclear extract shows that TNF-α stimulated HeLa nuclear extract has higher affinity to Ig-κB DNA than non-TNF-stimulated HeLa nuclear extract (4-h serum response). Therefore, the molecular binding scheme provides a rapid, quantitative, high throughput, and automated measurement of the DNA-binding protein NF-κB at low cost, which is beneficial for automated drug screening systems.https://www.mdpi.com/2079-6374/8/4/126biosensorFRETnuclear factorDNAfluorophore |
| spellingShingle | Zhaohui Wang Pak Kin Wong Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B Biosensors biosensor FRET nuclear factor DNA fluorophore |
| title | Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B |
| title_full | Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B |
| title_fullStr | Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B |
| title_full_unstemmed | Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B |
| title_short | Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B |
| title_sort | development of dna pair biosensor for quantization of nuclear factor kappa b |
| topic | biosensor FRET nuclear factor DNA fluorophore |
| url | https://www.mdpi.com/2079-6374/8/4/126 |
| work_keys_str_mv | AT zhaohuiwang developmentofdnapairbiosensorforquantizationofnuclearfactorkappab AT pakkinwong developmentofdnapairbiosensorforquantizationofnuclearfactorkappab |