Team:Bielefeld-CeBiTec/Results/it-really-works

A combined cell-free biosensor

It LITERALLY works on paper!

Project aim: Achieved!

Our measurement prototype for easy and fast evaluation of fluoresence emission

Nothing feels better than successfully bringing all aspects of a project together.

We achieved a lot during this summer, just take a look at this (far from complete) list to get an idea:

1. Highly efficient and robust cell-free synthesis of sfGFP on simple paper that can be stored
2. Cloning and characterization of multiple heavy metal sensors
3. Cloning and characterization of a date rape drug sensor
4. Establishment of a smartphone based fluorescence detection method
5. Implementing of an app to easily evaluate the measured fluorescence signals.

We consider our cell-free biosensor an outstanding instrument for the detection of harmful substances in liquids.

We see great need for a storable, transportable and easy-to-use water quality test strip all around the world.

Our development can help to improve the quality of living by revealing the quality of water.

We would like to demonstrate you in five steps how our final application works.

1. At first, you have to combine all reagents that are necessary to conduct cell-free protein synthesis. We made a simple cost estimation for the chemicals needed: To perform one of our 15 µL reactions you only have to spend 16 ¢.
2. When all compounds are combined, the reaction is applied on paper and freeze-dried. This procedure prevents the reaction from premature starting. Additionally, all remaining cells are killed.
3. The paper with the reaction is storable and transportable. For examination of water quality, you can simply add a water drop to the paper.
4. In no time you can take a picture of the paper with your smartphone. A filter combination enables you to capture the fluorescence that is emitted by the reporter protein
5. Our app evaluates the data and tells you if your water sample is contaminated.

In this way, we made it possible to detect various heavy metals. We proved this by using mercury contaminated water for rehydration of two different setups: One was our standard cell-free reaction setup and the other was our mercury detector. We took a picture, and our app immediatly gave us the result: "Your water is contaminated with: Mercury"!

Paper discs contatining the cell-free standards and Hg sensor, pictured without filters.

Cell-free biosensor for mercury detection in water samples. While the standard setup is strongly inhibited by Hg²⁺, the sensor setup strongly reacts when the metal is present.

During the reaction we had measured the fluorescence in a plate reader. In the nearby figures you can see: The standard CFPS setup performed bad when mercury was present, HOWEVER, when using our mercury detection setup, cell-free synthesis of our reporter protein sfGFP was greatly enhanced!

Water containing 6 µg/L Hg²⁺ strongly inhibits sfGFP expression in our standard cell-free setup. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.

Water containing 6 µg/L Hg²⁺ strongly enhances sfGFP expression in our cell-free mercury detection setup. High fluorescence signal can be measured after 60 min. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.

At molecular level, our biosensors work on basis of one simple concept: The expression of a gene is regulated by a protein. In case of a transcriptional repressor, the reporter protein is not expressed unless a particular analyte is present. In case of a transcriptional activator, expression of the reporter protein is enhanced in presence of the analyte.

This regulation is a central process in nature. Because our system uses this concept, it is highly extensible and can be used to tackle various real world problems.

We realized that date rape drug intoxications are currently one of these problems. With our development we introduce an easy and unprecedented date rape drug ingredient biosensor! To detect the date rape drug ingredient γ-hydroxybutyrate (GHB), we had to adjust only two components of our setup: The repressor protein and the DNA sequence that the protein recognizes.

Water containing 1% (v/v) of date rape drug ingredient GHB inhibits sfGFP expression in our standard cell-free setup. Data were acquired with Tecan plate reader. Error bars represent standard deviation of triplicates.

Detection of date rape drug ingredient GHB with our cell-free biosensor. Data were acquired with Tecan plate reader and normalized (for details see date rape drug result page). Error bars represent standard deviation of triplicates.

View the parts we build for our project.