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

Date Published: March 6, 2019

Publisher: Public Library of Science

Author(s): Alexander Gräwe, Anna Dreyer, Tobias Vornholt, Ursela Barteczko, Luzia Buchholz, Gila Drews, Uyen Linh Ho, Marta Eva Jackowski, Melissa Kracht, Janina Lüders, Tore Bleckwehl, Lukas Rositzka, Matthias Ruwe, Manuel Wittchen, Petra Lutter, Kristian Müller, Jörn Kalinowski, Hans-Joachim Wieden.

http://doi.org/10.1371/journal.pone.0210940

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.

Partial Text

Water quality assessment is an issue of global relevance. In regions that suffer from natural disasters or industrial accidents, for example, a quick and reliable test of water quality is indispensable to inhibit the spreading of diseases and to prevent human intoxications. Among the most widespread detrimental substances in water are heavy metals, in particular arsenic and mercury [1]. Mercury affects liver, kidney, and the central nervous system and may lead to severe diseases of these organs [2]. Mercury contamination of water causes steep health and human costs, as illustrated by the Minamata incidence, which lead to thousands of cases of methylmercury poisoning [3]. Hence, the detection of detrimental substances is a serious challenge of public health and environmental protection.

Homemade E. coli cell extract was established for paper-based applications. Cell-free protein synthesis of the reporter protein sfGFP worked well on paper when homemade extract was employed. The design of the genetic template was found to be of high importance to minimize transcription and translation limitations. The in vitro protein synthesis platform can be readily used for many applications, including synthetic gene networks [54], synthesis of proteins with non-natural amino acids [22], screening of enzymes [55] and others.

 

Source:

http://doi.org/10.1371/journal.pone.0210940

 

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