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Biosensors 2018, 8(4), 126; https://doi.org/10.3390/bios8040126

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

1,2,*
and
2,3
1
Department of Electrical Engineering and Computer Science, Texas A&M University-Kingsville, Kingsville, TX 78363, USA
2
Department of Aerospace & Mechanical Engineering, University of Arizona, Tucson, AZ 85721, USA
3
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)
Full-Text   |   PDF [1081 KB, uploaded 10 December 2018]   |  

Abstract

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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