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Biosensors 2018, 8(4), 126;

Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B

Department of Electrical Engineering and Computer Science, Texas A&M University-Kingsville, Kingsville, TX 78363, USA
Department of Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA
Biomedical Engineering and Mechanical Engineering, Department of Surgery, College of Medicine, Pennsylvania State University, University Park, PA 16802, USA
Author to whom correspondence should be addressed.
Received: 9 November 2018 / Revised: 29 November 2018 / Accepted: 5 December 2018 / Published: 10 December 2018
(This article belongs to the Special Issue FRET-Based Biosensors)
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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. View Full-Text
Keywords: biosensor; FRET; nuclear factor; DNA; fluorophore biosensor; FRET; nuclear factor; DNA; fluorophore

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Wang, Z.; Wong, P.K. Development of DNA Pair Biosensor for Quantization of Nuclear Factor Kappa B. Biosensors 2018, 8, 126.

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