Biosensors can be used to detect toxic substances in a highly specific and sensitive manner. Furthermore, they can be cheaper and easier to handle than conventional detection methods (Kaur et al. 2015). For this reason, they are a field of active research and a popular topic in the iGEM competition. Most of these biosensors make use of living microorganisms, which raises a number of problems. Among them are legal issues and safety concerns, as it must be prevented that genetically modified organisms are released into the environment. Moreover, these sensors are often not very user-friendly, as they have a limited shelf life and their application outside the laboratory is complicated. In order to solve these problems and thus make biosensors applicable in everyday life, we developed cell-free biosensors that can be used as paper-based test strips.

Our principal approach towards this end relies on cell-free protein synthesis (CFPS). This system is suitable for classical repressor- or activator-based biosensors, but by carrying out the expression of the reporter protein in a cell extract, biosafety problems are prevented. Furthermore, the cell extract remains functional when it is applied onto paper, and after lyophilization, such a test strip can be stored for a long time (Pardee et al. 2014).

We applied this system to the detection of heavy metals. Heavy metal contaminations are a global problem and a simple and cheap test would be a great help to many people. Several iGEM teams have dealt with the detection of heavy metals, so we decided to build upon this work and combine several heavy metal biosensors into one cell-free test strip. Consequently, we tested the following biosensors by previous iGEM teams, improved their characterization and optimized them for CFPS:

• arsenic (Edinburgh 2006, Buenos Aires 2013)
• mercury (Peking 2010, BBa_K346002 )

  The mercury sensor is already good characterized in vivo. We improved it by doing an in vitro characterization for it, which enables a use in cell free biosensor systems. Our data suggest, that the in vitro version, our sensor, indicates to the possibility to detect even lower concentrations.

• nickel (LMU Munich 2011,BBa_K549001 )
• lead (Brown 2007,BBa_I721003 )
• chromium (BIT 2013,BBa_K1058007 )

Nickel (BBa_K1758353) lead (BBa_K1758333) and chromium (BBa_K1758313) sensors were improved by characterization in vivo as well as in vitro.

In addition, we constructed a new heavy metal biosensor for copper BBa_K1758324.

In order to optimize our CFPS biosensors, we created a model that describes a repressor-based biosensor in our cell-free protein synthesis system. This model can help in adjusting the detection limit of our biosensors by predicting the necessary amount of repressor. As a result, test strips can be built that tell the user whether the concentration of a substance exceeds the safety limit.

In addition to heavy metals, we also wanted to tackle a problem that is currently very relevant in our area: The use of date rape drugs. These drugs are usually a mixture of different substances that produce narcotic effects and thus make the victim defenseless. By now, the detection of date rape drugs is only possible in retrospect in the laboratory and only in a limited time frame. There is no possibility to check suspicious drinks quickly and easily.

A biosensor based on cell-free protein synthesis produces a signal within one hour. However, for the detection of date rape drugs, the sensor needs to be even faster. In order to take this requirement into account, we devised a second biosensor design. This approach, called Plasmid Repressor Interaction Assay (PRIA), utilizes the interaction between a purified repressor protein and its operator sequence. On of these two components is immobilized on paper and the other carries a fluorescent label. In the presence of an analyte, the partners separate, which results in a loss of fluorescence at the specific spot.

Both CFPS and PRIA make use of fluorescence as an output signal because this requires no cofactors or subtrates and offers a good sensitivity (Daunert et al. 2000). However, fluorescence can usually not be seen with the naked eye. Nevertheless, it is possible to easily visualize fluorescence using a smartphone and filters. In 2014, iGEM Aachen have demonstrated this possibility using a filter for excitation. We improved this idea with a second filter in front of the camera, which enables everyone to specifally detect fluorescence by taking an image with their smartphone.

On top of that, we developed an app which automatically interprets the image and provides the user with information about the substances that were detected.

When developing a biosensor for date rape drugs, we wondered whether all the information we had gathered should be published, as some might contain the potential of misuse. In addition, we and other teams provide knowledge on our wikis in an open source context, that might contain misuse potential. This inspired us to deal with the Dual use issue in general. In our report we address legal and ethical questions. We analyze the position of iGEM as potential role model in the process of establishing awareness of the issue in the scientific community. While iGEM does not yet provide guidelines and security risk assessment, we propose to integrate the issue in the biosafety page and forms to complete the already existing safety measures. The 28 pages report is provided as PDF and summarized on our wiki.

Let´s take a closer look at CFPS!