Development of small molecule biosensors by coupling the recognition of the bacterial allosteric transcription factor with isothermal strand displacement amplification

Abstract

Here, we demonstrate an easy-to-implement and general biosensing strategy by coupling the small-molecule recognition of the bacterial allosteric transcription factor (aTF) with isothermal strand displacement amplification (SDA) in vitro. Based on this strategy, we developed two biosensors for the detection of an antiseptic, p-hydroxybenzoic acid, and a disease marker, uric acid, using bacterial aTF HosA and HucR, respectively, highlighting the great potential of this strategy for the development of small-molecule biosensors.

An integrated-molecular-beacon based multiple exponential strand displacement amplification strategy for ultrasensitive detection of DNA methyltransferase activity

“…Similarly, using I t / I 0 as a criterion, the concentration of primer 1 was optimized at 100 nM (Fig. S2†), and the amounts of KFP and Nb.BbvcI were selected at 0.5 and 1 units in a total volume of 100 μL, according to previous research 35…”

Development of a Biosensor for Detection of Benzoic Acid Derivatives in Saccharomyces cerevisiae

“…We also verified that the measured fluorescent signal is proportional to the quantity of mCitrine measured by Western Blot (Supplementary Figure 1). Notwithstanding the fact that a previous in vitro study described a sTF activated by pHBA (Yao et al, 2018), to our knowledge, sHbaR is the first biosensor that can be activated by pHBA in vivo, in S. cerevisiae.…”

A Förster Resonance Energy Transfer‐Based Ratiometric Sensor with the Allosteric Transcription Factor TetR

“…Only very recently have aTFs been examined as biomolecular recognition elements for in vitro sensing. [ 10,28–31 ] Of the limited number of signal transduction mechanisms explored, only ours utilizes FRET. One homogeneous assay achieved nanomolar sensing using an amplified luminescent proximity homogeneous assay (AlphaScreen).…”

“…[ 28 ] However, this nonratiometric approach requires specialized equipment (a plate reader equipped with a 680 nm laser), commercialized beads that are expensive, and unstable under visible light, and provides a negative output signal. [ 28,31 ] Two other assay designs provide only an indirect measure of aTF–DNA binding by amplifying and quantifying (un)bound DNA. [ 29–31 ] Using existing methods of DNA detection facilitates incorporation into existing workflows but lacks the advantages of a real‐time, homogeneous assay without substantial gains in the LOD or linear dynamic range.…”

“…[ 28,31 ] Two other assay designs provide only an indirect measure of aTF–DNA binding by amplifying and quantifying (un)bound DNA. [ 29–31 ] Using existing methods of DNA detection facilitates incorporation into existing workflows but lacks the advantages of a real‐time, homogeneous assay without substantial gains in the LOD or linear dynamic range. [ 29–31 ] Our FRET‐based sensor maintains the rapidity and convenience of the homogeneous assay, while incorporating a ratiometric output and widely accessible fluorophores to facilitate broader adoption.…”