Building a biosensor like playing with toy bricks?

Biosensors that are built on a modular principle with interchangeable synthetic components open up fascinating new approaches in imaging cellular processes.

June 28, 2018

Thousands of different molecules are involved in ‘keeping the engine running’ in each of our cells and thousands more make up the ‘machinery of life’. Detecting and imaging all the processes involved requires a great variety of tools. Scientists at the Max Planck Institute for Medical Research in Heidelberg and the École Polytechnique Fédérale de Lausanne have now developed a new class of semisynthetic biosensors for measuring the concentration of nicotinamide adenine dinucleotide phosphate (NADPH/NADP⁺), a molecule that e.g. relieves ‘our stress’. Moreover, the modular approach behind this new system will be applicable in a much wider range of measurements.

The sensor protein is labeled via SNAP-tag with a synthetic molecule containing a FRET donor (green star) and a SPR inhibitor (blue ball, SMX), and via Halo-tag with a FRET acceptor. NADPH (orange ball) and NADP+ (purple ball) compete for the cofactor-binding site of SPR. The sensor can monitor NADPH/NADP+ ratio changes by switching from a closed conformation to an open conformation, with high and low FRET efficiency, respectively.

The sensor protein is labeled via SNAP-tag with a synthetic molecule containing a FRET donor (green star) and a SPR inhibitor (blue ball, SMX), and via Halo-tag with a FRET acceptor. NADPH (orange ball) and NADP+ (purple ball) compete for the cofactor-binding site of SPR. The sensor can monitor NADPH/NADP+ ratio changes by switching from a closed conformation to an open conformation, with high and low FRET efficiency, respectively.

“Integrating a variety of synthetic components into the sensor is the key element of this approach”, says Fabio Raith, one of the authors of the paper and a PhD student in the department of Prof. Kai Johnsson (Chemical Biology). This rational design principle enables the scientists to change the characteristics of the sensors and thus engineer its properties to a new degree. The sensors can be adapted to different environmental conditions and tuned to emit different colors. This is a necessary step above all for the practical use of the sensors in living tissue or for detection in blood samples.

Previous approaches to measuring NAD⁺were ‘protein-only’: they used no synthetic components. The new class of biosensors is based on a system widely used in Kai Johnsson’s department – the Snifit system. This system is based on a set of standard components: a specific selection of these are combined so as to ‘shine’ when they bind to the target in question, and be detected with a camera. The authors of the paper have now expanded the Snifit system by introducing synthetic components that can be designed more easily and modified rapidly for new targets.

Time course of the FRET ratio (TMR/FRET) of cytosolic NADP-Snifit upon perfusion of sulfapyridine and increasing concentration of H2O2 to observe how cells react to oxidative stress.

Time course of the FRET ratio (TMR/FRET) of cytosolic NADP-Snifit upon perfusion of sulfapyridine and increasing concentration of H2O2 to observe how cells react to oxidative stress.

“What really fascinates and excites me is that the sensor can be made specific for a different molecule just by introducing two mutations”, says Fabio Raith further. The system could therefore be used for a wide variety of molecules with little effort and very fast. In this way they have already been able to use the sensor in living cells to detect the two different cofactors: free NAD⁺ and NADPH/NADP⁺.

These molecules play essential roles in various cellular processes regulating energy metabolism, biosynthesis and the defense against oxidative stress. For example, when we get a suntan, we are exposed to sunlight and therefore UV radiation. This promotes the building of oxygen species that harm our cells. Using the biosensors, scientists from the two institutes have now been able to monitor the influence on these toxic molecules on NADPH/NADP⁺ levels and demonstrate the remarkable ability of our body’s cells to regenerate after such a stress situation.