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.

Figure 1

The structure-switching electrochemical TF sensor is based on the use of a redox modified DNA probe, which is in equilibrium between two conformations (non-binding, left, and binding, right). Binding of the TF to its consensus sequence, shown in red, shifts the population towards binding conformation, placing the methylene blue redox tag close to the electrode surface and thus increasing its electron transfer rate (eT). Thus, in the presence of the target TF, TATA-binding protein (TBP), a robust current signal increase is observed at the redox potential characteristic of methylene blue.

Figure 2

A microfluidic sample chamber containing two sensors supports the detailed measurement of the TF TBP binding in ~10 ul samples. a, the microfluidic chips used in this study. b, the dose-response behavior of the electrochemical TF sensor to the protein TBP is robust and sensitive in buffer or in 250 μg/ml HeLa nuclear extract (NE), displaying an affinity of 121 ± 20 nM. c, Endogenous TBP levels in nuclear extract can be quantitatively obtained using Eq. 1, comparing sensor response in extract to response in pure buffer (S_min, baseline signal) and in the presence of saturating levels of TBP (S_max, maximum response). NE analyzed by this method was shown to contain 4 ± 2 nM of TBP.