A paper-based, cell-free biosensor system for the detection of heavy metals and date rape drugs

Abstract

Biosensors have emerged as a valuable tool with high specificity and sensitivity for fast and reliable detection of hazardous substances in drinking water. Numerous substances have been addressed using synthetic biology approaches. However, many proposed biosensors are based on living, genetically modified organisms and are therefore limited in shelf life, usability and biosafety. We addressed these issues by the construction of an extensible, cell-free biosensor. Storage is possible through freeze drying on paper. Following the addition of an aqueous sample, a highly efficient cell-free protein synthesis (CFPS) reaction is initiated. Specific allosteric transcription factors modulate the expression of ‘superfolder’ green fluorescent protein (sfGFP) depending on the presence of the substance of interest. The resulting fluorescence intensities are analyzed with a conventional smartphone accompanied by simple and cheap light filters. An ordinary differential equitation (ODE) model of the biosensors was developed, which enabled prediction and optimization of performance. With an optimized cell-free biosensor based on the Shigella flexneri MerR transcriptional activator, detection of 6 μg/L Hg(II) ions in water was achieved. Furthermore, a completely new biosensor for the detection of gamma-hydroxybutyrate (GHB), a substance used as date-rape drug, was established by employing the naturally occurring transcriptional repressor BlcR from Agrobacterium tumefaciens .

The Evolution of Cell Free Biomanufacturing

“…These systems have been deployed to detect pathogens such as norovirus [68], Ebola virus [69] and Zika virus [70]. In addition, initial studies have showed that paper-based cell free sensors can detect the presence of heavy metals such as mercury and drugs such as γ-hydroxybutyrate, by utilizing the transcriptional regulators, MerR and BlcR, respectively [71]. The portability offered by these systems further underscores their usefulness in the field.…”

Design and optimization of a cell-free atrazine biosensor

“…1 However, previously reported cell-free biosensors have so far been limited to detecting either nucleic acids 2,3 or chemical contaminants that can be directly sensed with well-characterized allosteric protein transcription factors or riboswitches. 8,9,11–13…”

TXO: Transcription-Only genetic circuits as a novel cell-free approach for Synthetic Biology

“…Previous works 212 with RNA aptamers for metal sensing, DasGupta et al mentioned the use of a truncated 213 form of the Spinach aptamer as a Pb 2+ sensor, but this could have lead to false results 214 at concentrations <2 ppm due to a lack of sensitivity and a weak output response [30]. 215 Since TXO systems are considerably simpler than TX-TL systems, with no require-216 ment for ribosomes, tRNA, or associated enzymes, we expect that it will be possible 217 to freeze-dry such systems for storage and distribution as previously reported for TX-218 TL systems [8,28]. Fluorescent signals may be further strengthened using alternative 219 fluorophores such as DHFBI-1T [29].…”

“…For instance, Pardee and colleagues [7] developed a cell-free paper toehold-switch sensor 22 for detection of the Zika virus that takes hours for the final visual results to come out [6]. 23 Paper-based cell-free sensors that detected heavy metals and gamma-hydroxybutyrate 24 by expressing sfGFP needed at least one hour for producing a measurable response [8]. 25 Considering the need for in-situ field analysis, an improvement of cell-free systems 26 response time is necessary [9,10].…”

“…Paper-based cell-free sensors that detected heavy metals and gamma-hydroxybutyrate 24 by expressing sfGFP needed at least one hour for producing a measurable response [8]. 25 Considering the need for in-situ field analysis, an improvement of cell-free systems 26 response time is necessary [9,10].…”