2D Biosensor

With our 2D biosensor technology we are able to detect the pathogen Pseudomonas aeruginosa on solid surfaces. The sensor system is comprised of two distinct but inseparable modules, a biological part and a technical part:

• Sensing chips containing Cellocks, our engineered detective cells that fluoresce in the presence of the pathogen, make up the biological part of Cellock Holmes.
• Our measurement device WatsOn and the complementary softare Measurarty complete our sensing technology on the technical side.

Principle of Operation

Cellock Holmes is devised based upon a SynBio approach comprised of a two-dimensional biosensor and a measurement device. The two-dimensional biosensor is designed to recognize quorum sensing molecules secreted by the pathogen cells and to generate a distinct fluorescence signal, while the measurement device is designed to recognize and analyse the produced signal. To overcome the limitation of our REACh construct to bacteria that secrete autoinducers, we developed an alternative detection method (Galectin-3 construct), which is based on tagging cells with a fluorescent reporter.

[graph quorum senising]

Our sensor cells are immobilized in agar chips. To make the chips, we mix the sensing cells, also known as Cellocks, with liquid LB agar. In the course of our project, we designed a casting mold specifically for the production of our agar chips. When the agar has cooled down, the chips are cut out of the mold and are ready to use. Storage of the readily usable sensor chips is possible for 2 days at 4 °C when using LB medium or for 5 days if TB-medium is used. A detailed description of the sensor chip manufacturing can be found in our Protocols section.

Application of WatsOn for investigation of solid surfaces.

The application of Cellock Holmes for detection of P. aeruginosa cells is simple: fist, a sampling chip is placed on a hard surface that is potentially contaminated with the pathogen. Second, the sampling chip is removed from the surface and put onto one of our sensor chips. Theorectically the sensor chips could be directly used for sampling, but this was avoided in our project to match biosafety regulations. Third, the two layered chip-stack is put into a petri dish, which is inserted into our measurement device WatsOn for evalutation.

Mode of action inside WatsOn.

Inside WatsOn the chips are incubated at 37 °C and populations of microorganisms on the sampling chip start to multiply. P. aeruginosa secrets an increasing number of 3-oxo-C₁₂ homoserine lactones (HSLs) when multiplying. These HSLs induce the generation of a fluorescent signal in our sensor cells, which is described in more detail in the REACh Construct section. The chips can be illuminated with blue light at any time, while WatsOn takes a picture of the chip. The software Measurarty then analyzes any fluorescent signal. Depending on the intensity of the signal and the size of the spot, Cellock Holmes can calculate concentration and distribution of P. aeruginosa on the sampled surface.

Our achievements so far

Medium

Prior to using our own device for detection of fluorescence in our sensor chips we used equipment readily available in the lab: a Molecular Imager® Gel Doc^TM XR+ from BIO-RAD. The Gel Doc^TM uses UV light and has only two filter. Because of this it was not ideal for our project. We could see iLOV but no GFP (iLOV_GFP_HM_1,5h.png) LB medium has under UV light a high fluorescence. The background fluorescence was to high to see a signal of our cells so we used minimal media (NA, M9, Hartman) to minimize the background. This worked fine for the first tries but the cells don't grow well in the minimal media. (Chip_medium_geldoc.png) There was the idea to store the chips at -20 °C, ready for use. But the cells died. Adding 5-10% glycerol resulted in an expression stop of fluorescence proteins. So the idea was rejected. In our own device WatsOn with the optimized light (450/ 480 nm) the LB showed no fluorescence. The cells grow better there and so we use this now. For longer storage up to 5 days in the fridge (+ 4°C) TB medium can be used. It has a smal background fluorescence in WatsOn. (5Tage_K131026_neb_tb_1,5h)

Agar concentration

For the sensor chip an agarose concentration of 1.5% is used. We first tried agar. When the agar concentration is lower than 1.5% the chip is not stable and brakes fast. When the agar concentration is higher than 1.5% the medium gets solid before pouring it into chip form. In the end we choose agarose instead of agar because the agarose is better linked and so the diffusion is not so high.

Chip form

First we wanted to produce every chip individually. For a plane surface we wanted to use microscope slides. But the agar was to liquid and leaked. After that we made a closed form in which you can give the ager with a pipette. But this chips had big bubbles and were really difficult to produce. (2_chipform.jpg) In the end we use a self made open form. Here you produce 9 chips at the same time. You just have to cut them out (anhand) the lasered lines. This is a fast, easy and efficient way to produce chips. And because of the surface tension the chips are plane. (final_chipform.jpg)

Induction

For the induction of the used constructs we use IPTG or 3-oxo-C12 HSL. The sensor cells with K1319042 in BL21 can detect a IPTG concentration of 1 mM (0,2 µl). (2µl_IPTG_1mM_K1319042_1h.png) The sensor with the REACh constructs in BL21 can detect a IPTG concentration of ??? (Plate reader???) The sensor cells with K131026 in BL21 can detect an HSL concentration of 500 µg/ml (0,2 µl). It also can detect Pseudomonas aeruginosa. (Zeitaufnahmen bearbeitet von Arne)

Testing our sensor Chips with a Platereader

Detecting the 3-oxo-C₁₂ HSL with K131026 in our sensor chip with WatsOn.

Detecting Pseudomonas aeruginosa with K131026 in our sensor chip with WatsOn.