Quantification of Transcription Factor Binding in Cell Extracts Using an Electrochemical, Structure-Switching Biosensor

Abstract

Transcription factor expression levels, which sensitively reflect cellular development and disease state, are typically monitored via cumbersome, reagent-intensive assays that require relatively large quantities of cells. Here we demonstrate a simple, quantitative approach to their detection based on a simple, electrochemical sensing platform. This sensor sensitively and quantitatively detects its target transcription factor in complex media (e.g., 250 μg/ml crude nuclear extracts) in a convenient, low-reagent process requiring only 10 μl of sample. Our approach thus appears a promising means of monitoring transcription factor levels.

Maximizing the Signal Gain of Electrochemical-DNA Sensors

“…1 Originally used for the detection of DNA 2–9 and RNA 10 targets, the platform has since been expanded to the detection of a wide range of small molecules, 11,12 inorganic ions, 13,14 and proteins, 12,15–17 including antibodies, 18,19 via the introduction of aptamers and nucleic-acid-small molecule and nucleic-acid-peptide conjugates as recognition elements (reviewed in refs 20 and 21). Irrespective of their specific target, all of these sensors are predicated on a common mechanism: binding alters the efficiency with which the attached redox reporter approaches the electrode due to either the steric bulk of the target or the changes in the conformation of the probe.…”

Sensitive detection of transcription factors in cell nuclear extracts by using a molecular beacons based amplification strategy

“…S1) using the MB probe without Nb.BbvCI amplification, the sensitivity of which was more than 2 orders of magnitude poorer than that of the Nb.BbvCI amplified method. Notably, the sensitivity of the proposed method had improved by as much as 3 orders of magnitude as compared with the previously reported E-DNA sensor based electrochemical assay (Bonham et al, 2012) and up to 4 orders of magnitude as compared with FRET-based assay as well (Heyduk and Heyduk, 2002). More importantly, to compare with electrophoretic mobility shift assay (EMSA), an established standard assay, our method has a high sensitivity than EMSA (0.25 g L À 1 , 3.8 mM) for the detection of NF-κB p50 (Renard et al, 2001).…”

“…Owing to the pivotal role of TFs in gene expression as well as its close relationship with human diseases, the accurate measurement of transcription factors is of great importance to both medical diagnosis and biological research (Helin, 1998). Recent years have seen the development of a broad class of biosensors for the assay TFs (Bonham et al, 2012;Vallée-Bélisle et al, 2011;Yin et al, 2014;Zhang et al, 2012). Unfortunately, these methods for monitoring TF concentration or binding activity are generally cumbersome and timeconsuming.…”

Nanostructured gold dsDNA sensor for early detection of breast cancer by beta protein 1 (BP1)

“…Most nano-gold (NG) sensors modified with single-stranded DNA are designed to characterize the hybridization between the two complimentary strands which allows electrochemical charge transport [14,20,24–27]. A series of conjugated charge donors or acceptors are employed as redox active probes interacting by π-stacked base pairs in dsDNA to monitor the electrochemical binding event [21,24,28–34].…”