Dedicated and highly efficient EPR analysis of small volume samples in a range of few µl is now made simple with a novel device invented at the Weizmann Institute of Science. This device features a new ejection mechanism and a unique cold trap which enables collection of all time points in a RFQ series in one continuous experiment.
In order to design and develop inhibitors for therapeutic purposes, the reaction mechanisms of enzymes must be understood. For biological applications, a common methodology of addressing this need is combining Rapid Freeze Quench with Electron Paramagnetic Resonance (RFQ)-EPR, which allows the trapping and analysis of short lived intermediates in chemical reactions. However, commercial RFQ-EPR devices are limited for high field EPR applications due to the demand of large sample volumes for each time point.
Prof. Goldfarb and her team built a new RFQ apparatus based on microfluidic flow and unique ejection and freezing systems, which can be used for collecting small volume samples in capillaries for high field EPR.
This technology, combined with the variety of W-band high resolution EPR technique (ENDOR, DEER and ESEEM) enables better mechanistic studies of enzymatic reactions, with an emphasis on structural transformations during the reaction, in an efficient and accurate way.
- Collecting all RFQ time points in one continues experiment.
- Produce small volume samples in the range of a few µl, and handles small capillaries, for high field ERP.
- An improved dead time of ~5ms, relative to the commercial RFQs with a typical dead-time of 510 ms.
- Ease-of-use and speed.
The innovative apparatus consists of two main parts: the microfluidic device and the freeze-quench setup. The microfluidic device comprises a mixer, which mixes the two reacting solutions, a flow path where the reaction occurs, and a sprinkler from which the solution is sprayed out of the device. Prof. Goldfarb and her colleagues improved the common mixing device by adding a fast stream of nitrogen gas which mixes with the ejected reaction solution, and sprays the frozen aerosol out in tiny drops at high speed.
The innovative RFQ device was planned to have a cold solid surface on which the freezing happens rather than the traditional ejection into a cold liquid, in order to minimize the losses of the frozen solution. Moreover the plate rotates at a speed correlated to the flow speed of the solution, thus samples of different reaction times can freeze on a different radius. The frozen samples are then collected into quartz capillaries.