American researchers have developed a probe for marking biomolecules that begins to fluoresce only when it is “switched on” by binding. As reported in the journal Angewandte Chemie, the reaction takes place very quickly and the difference in brightness between the “on” and “off” states is two orders of magnitude bigger than for conventional activatable probes.
Marking biomolecules in living cells with fluorescent probes is a well-established technique. New research possibilities open up when these probes are combined with bioorthogonal reactions. Such reactions can occur inside a living system without disrupting normal biochemical processes.
This makes it possible to generate “turn-on” probes: a bioorthogonal reaction binding partner is bound to the biomolecule of interest (without affecting it) and acts as an anchoring site for the fluorescent probe. The probe is devised so that its fluorescence is significantly increased when it binds to the anchoring site. Because the probes not bound to the target fluoresce far less, background fluorescence is reduced. This eliminates the need for complex washing procedures that delay observation of the cells.
For all of this to work, the probe system must work without a toxic catalyst, react quickly to allow for time-resolved observation of biological processes, and fluoresce very strongly after being “turned on” to maximize the signal–strength relative to the background. It has not previously been possible to meet all of these requirements in one system.
A team led by Ralph Weissleder at Massachusetts General Hospital and Harvard University has now developed a system that fits the bill: an unusually bright, fast reacting, biocompatible probe system with a large difference between the switched on and switched off states.
The new probe consists of two components: The first is a fluorescent dye called BODIPY (boron dipyrromethene), a three-ring system with a subunit made of one boron, two nitrogen, and two fluorine atoms. The second component is a tetrazine molecule, a six-membered ring containing four nitrogen and two carbon atoms. Tetrazine quenches the fluorescence of BODIPY, which passes incoming energy off to the tetrazine component without radiation instead of fluorescing.
The reaction destroys the probe’s tetrazine group, turning off the quenching of the fluorescence and allowing the BODIPY molecule to glow an intense green. The researchers recorded fluorescence over a thousand times stronger than that of the probe in the “off” state. This is two orders of magnitude stronger than all previously described turn-on probes.
The success of this system is due to the particularly strong fluorescence quenching made possible by the special electronic constellation and spatial arrangement of the BODIPY and tetrazine components relative to each other.About the Author
Ralph Weissleder | Angewandte Chemie
27.03.2015 | Oak Ridge National Laboratory
How did the chicken cross the sea?
27.03.2015 | Michigan State University
In an experiment at the Department of Energy's SLAC National Accelerator Laboratory, scientists precisely measured the temperature and structure of aluminum as...
The IPH presents a solution at HANNOVER MESSE 2015 to make ship traffic more reliable while decreasing the maintenance costs at the same time. In cooperation with project partners, the research institute from Hannover, Germany, has developed a sensor system which continuously monitors the condition of the marine gearbox, thus preventing breakdowns. Special feature: the monitoring system works wirelessly and energy-autonomously. The required electrical power is generated where it is needed – directly at the sensor.
As well as cars need to be certified regularly (in Germany by the TÜV – Technical Inspection Association), ships need to be inspected – if the powertrain stops...
When an earthquake hits, the faster first responders can get to an impacted area, the more likely infrastructure--and lives--can be saved.
The Atlantic overturning is one of Earth’s most important heat transport systems, pumping warm water northwards and cold water southwards. Also known as the Gulf Stream system, it is responsible for the mild climate in northwestern Europe.
Scientists now found evidence for a slowdown of the overturning – multiple lines of observation suggest that in recent decades, the current system has been...
Because they are regularly subjected to heavy vehicle traffic, emissions, moisture and salt, above- and underground parking garages, as well as bridges, frequently experience large areas of corrosion. Most inspection systems to date have only been capable of inspecting smaller surface areas.
From April 13 to April 17 at the Hannover Messe (hall 2, exhibit booth C16), engineers from the Fraunhofer Institute for Nondestructive Testing IZFP will be...
25.03.2015 | Event News
19.03.2015 | Event News
17.03.2015 | Event News
27.03.2015 | Agricultural and Forestry Science
27.03.2015 | Materials Sciences
27.03.2015 | Ecology, The Environment and Conservation