title: Electron spin resonance scanning probe spectroscopy for ultrasensitive biochemical studies.
abstract: Electron spin resonance (ESR) spectroscopy's affinity for detecting paramagnetic free radicals, or spins, has been increasingly employed to examine a large variety of biochemical interactions. Such paramagnetic species are broadly found in nature and can be intrinsic (defects in solid-state materials systems, electron/hole pairs, stable radicals in proteins) or, more often, purposefully introduced into the material of interest (doping/attachment of paramagnetic spin labels to biomolecules of interest). Using ESR to trace the reactionary path of paramagnetic spins or spin-active proxy molecules provides detailed information about the reaction's transient species and the label's local environment. For many biochemical systems, like those involving membrane proteins, synthesizing the necessary quantity of spin-labeled biomolecules (typically 50 pmol to 100 pmol) is quite challenging and often limits the possible biochemical reactions available for investigation. Quite simply, ESR is too insensitive. Here, we demonstrate an innovative approach that greatly enhances ESR's sensitivity (>20000× improvement) by developing a near-field, nonresonant, X-band ESR spectrometric method. Sensitivity improvement is confirmed via measurement of 140 amol of the most common nitroxide spin label in a ≈593 fL liquid cell at ambient temperature and pressure. This experimental approach eliminates many of the typical ESR sample restrictions imposed by conventional resonator-based ESR detection and renders the technique feasible for spatially resolved measurements on a wider variety of biochemical samples. Thus, our approach broadens the pool of possible biochemical and structural biology studies, as well as greatly enhances the analytical power of existing ESR applications.
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Jason P
authors: JP
authors: Campbell
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Jason T
authors: JT
authors: Ryan
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Pragya R
authors: PR
authors: Shrestha
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Zhanglong
authors: Z
authors: Liu
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Canute
authors: C
authors: Vaz
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Ji-Hong
authors: JH
authors: Kim
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.|§Department of Electrical and Computer Engineering, George Mason University, 4400 University Drive, Fairfax, Virginia 22030, United States.
authors: Vasileia
authors: V
authors: Georgiou
authors: †National Institute of Standards and Technology, 100 Bureau Drive, MS 8120, Gaithersburg, Maryland 20899, United States.
authors: Kin P
authors: KP
authors: Cheung
mesh_terms: D004578:Electron Spin Resonance Spectroscopy; D005609:Free Radicals / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis*; D008872:Microwaves
chemical_list: D005609:Free Radicals; D008565:Membrane Proteins