Biology subjects

Dujovne, M. V.

Publications and source records attributed to Dujovne, M. V..

2 recordsLinked to original sources

bioRxiv2025

Lateral flow cell-free transcriptional assay for contaminant detection

Cell free transcriptional biosensors are emerging as a powerful technology, offering enhanced capabilities for detecting chemical contaminants in settings where traditional analytical techniques fall short of societal needs. However, the sensitivity of these biosensors for many critical chemical contaminants often remains insufficient to meet regulatory detection limits, while maintaining portability and ensuring selectivity. In this work, an in vitro transcription (IVT) based lateral flow assay (LFA) is reported, which is 1000 times more sensitive than reported IVT portable detection methods. Through combining the amplification power of IVT with nucleic acid LFA-based avidity effects, we develop rapid, ultra-sensitive turn-on sensors. Our sensors meet regulatory detection limits for lead, a particularly challenging metal ion contaminant, in untreated spring water samples collected from a basin that exemplifies the environmental monitoring challenges. Additionally, we show that by optimizing the IVT conditions, we can tune the limit of detection to produce an on-off signal reaching the WHO regulation threshold and others. Finally, we evaluate the selectivity of different metal-responsive repressors in the context of in vitro biosensing and discuss how to identify potentially hazardous samples with an easy-to-use and point-of-care assay.

synthetic biology↗

bioRxiv2024

Engineering a cell-free biosensor signal amplification circuit with polymerase strand recycling

Cell-free systems are powerful synthetic biology technologies because of their ability to recapitulate sensing and gene expression without the complications of living cells. Cell-free systems can perform even more advanced functions when genetic circuits are incorporated as information processing components. Here we expand cell-free biosensing by engineering a highly specific isothermal signal amplification circuit called polymerase strand recycling (PSR) that leverages T7 RNA polymerase off-target transcription to recycle nucleic acid inputs within DNA strand displacement circuits. We develop design rules for PSR circuit components and use these rules to construct modular biosensors that can directly sense different RNA targets with limits of detection in the nM range and high specificity. We then use PSR for signal amplification within allosteric transcription factor-based biosensors for small molecule detection. We use a double equilibrium model of transcription factor:DNA and transcription factor:ligand binding interactions to predict biosensor sensitivity enhancement by PSR, and then demonstrate this approach experimentally by achieving 3.6-4.6-fold decreases in biosensor EC50 to sub micromolar ranges. We believe this work expands the current capabilities of cell-free circuits by incorporating PSR, which we anticipate will have a wide range of uses within biotechnology.

synthetic biology↗