Sensitivity Enhancement
BCMB 8190
References
• High Frequency Dynamic Nuclear Polarization, Ni, QZ…Griffin, RG,
Accounts of Chemical Research, 46:1933-1941, 2013.
• Dynamic Nuclear Polarization Surface Enhanced NMR Spectroscopy,
Rossini, Aaron J.; Zagdoun, Alexandre; Lelli, Moreno; et al., Accounts of
Chemical Research, 46:1942-1951, 2013.
• NMR of hyperpolarised probes, Witte, C; Schroeder, NMR in
Biomedicine, 26:788-802, 2013.
• Chemistry and biochemistry of C-13 hyperpolarized magnetic resonance
using dynamic nuclear polarization, Keshari, Kayvan R.; Wilson, David
M., Chemical Society Reviews, 43: 1627-1659, 2014.
• Principles and progress in multidimensional NMR, M. Mishkovsky and L.
Frydman, Ann. Rev. Phys. Chem. 60:429-448, 2000.
Sensitivity is the Major NMR Limitation:
How can it be improved?
• Better detection: cryo-probes, SQUIDS, mechanical
oscillators
• Higher magnetic fields: 23.4T (1.0 GHz)
• Higher polarization: low temp, transfer from systems with
higher , pumping
• Signal  3B02h2 / (162kT); P  B0h/(4kT)
What will higher sensitivity allow?
•
•
•
•
More efficient studies of biomolecules – solids and liquids
Studies of rapid reactions – metabolic flux
Observation of molecules on surfaces or in reactive states
In vivo studies – MRI/MRS
Increasing Polarization: Alkali Metal Spin Exchange
From: B.M. Goodson (2002) J. Mag. Reson. 155:157–216.
Depends on the use of circularly polarized light
and the conservation of angular momentum
Experimental Set-up: Optical Pumping
and Spin Exchange of Alkali Metal
1) 129Xe (or 3He) at low pressure (~ 8 atm) is enclosed in a cylindrical
glass chamber in a low magnetic field (~ 10 G). Trace amounts of Rb
added, heated to 200°C.
2) Circularly
polarized laser light
applied. λ = 794.8
nm, 5s to 5p (D1)
transition of Rb
3) Absorption of the laser light produces a high electronic polarization
in the Rb atoms by means of optical pumping. Polarization transferred
to 129Xe by flip-flop term of Fermi-contact hyperfine interaction. Can
reach 10% polarization – enhancements of 10,000
3He
Coronal HP
lung images of
patients with asthma
a) patient with mild disease
and normal spirometry (FEV1 =
98% of predicted) shows few
pleural-based peripheral
ventilation defects
b) patient with severe asthma
(FEV1 – 36% of predicted)
has large number of defects
Moller, H.E., et al., MRI of the lungs using hyperpolarized noble gases. Magn Reson Med, 2002. 47(6): p. 1029-51.
Fluxomics: Important Branch of Metabolomics
• Living systems are not at thermodynamic equilibrium
• They are a network of interacting biochemical reactions
• Flow of materials through this network can be as
diagnostic of disease as the elevation of a metabolite
• Flows can be very rapid
– Blood flows throughout the body on a time scales of 10 sec
– Mucosal cells can replace mucopolysaccharides in <30 min
– 108 Hepatoma cells in a bioreactor supplied with 4 mM
pyruvate can convert ~1/2 to lactate in ~300 sec
• This places high demand on methodology and sensitivity
of analytical methods
One Option: Dynamic Nuclear Polarization (DNP)
• Requires addition of a free radical and micro wave irradiation
• Transfers large electron polarization to nucleus (e / p = 650)
• Samples are cooled to ~2K for additional enhancement
m = -1/2
298K
2K
m = +1/2
1H/13C/15N
detection depends
on difference of spin state
populations – very small
2K
Microwave irradiation
transfers e- polarization
to nucleus
2K
m = +1/2
Larger moment for electrons
in added free radical means
larger energy gap and
population difference
Rapid dissolution and
slow spin relaxation
preserves polarization
For 298K observation
m = -1/2
Several Transfer Mechanisms:
• The Overhauser effect – liquids and certain solids
• The solid effect – solids with strong hyperfine coupling
• The cross effect – three spin process in solids at higher fields
• Thermal mixing - most probable for dissolution DNP
All depend on dipole-dipole interactions
between electron and nuclear spins
B0

r
’
r’
r’’
HD = (012h2)/(163r3)(A+B+C+D+E+F)
A = - Iz1Iz2(3cos2 - 1),
B = (1/4)(I+1I-2 + I-1I+2) (3cos2 - 1)
…
Useful References:
Maly T, et al, (2008) J. Chem. Phys. 128:52211-19; Smith AA, et al. (2012) J.
Chem. Phys. 136:015101; Jannin S, et al. (2011) Chem. Phys. Let. 517:234
Dynamic Nuclear Polarization (DNP)
the Overhauser Mechanism
• Illustrated for a 13C-electron pair
• Irradiate with micro waves
• Allow relaxation in which W0 is most efficient
 0
 
 
 +


 /2
 +/2

 0
 +/2

 /2
 /2+

 /2
 +
-/2
-
Enhancement is approximately ½ (e /C)  1300
-/2
-
Other Mechanisms:
Thermal Mixing (2&3):
• Requires interactions leading to broadening of ESR line to be comparable
to, or larger than, nuclear precession frequency
• Spin temperature in the rotating frame becomes equivalent for electrons
and nuclei (~10 mK)
• Polarization (nucleus) = tanh(h/2kBTS), ~0.1 for 13C, ~0.04 for 15N
Solid Effect (1):
• Requires that interactions between electron spin pairs and nuclearelectron spin pairs to be large – spin states are mixed
• Normally forbidden transitions ( >> ) are directly driven
• Enhancement  e/ n, max at  = e +/- n
Cross Effect (more recent high field solids):
• A 3 spin process (two electrons, one nucleus)
• Difference in electron frequency must be of
order of nuclear precession (e1 - e2 = +/- n)
• Enhancement  e/ n, max at  = e +/- n
• Sums dictate optimal microwave irradiation
e - n
e + n
Wind RA. et al. (1985) Prog. NMR Spec.,17: 33-67
Types of Radicals and Sample Preparation
4-amino-TEMPO
DBPA
Trityl Radical
OX63 Trityl Radical
~100 – 500 mM substrate, 15 mM trityl radical, in 100-400 L solvent
Solvents need to be glassy when frozen (glycerol/water, methanol/water, urea/water)
After 1-2 hrs polarization dissolution is with 2-4 mL heated buffer of choice
Enhancing NMR sensitivity to observe metabolism
Hyperpolarization detected by solution NMR increased 13C sensitivity 10,000x
(Ardenkjaer-Larsen,et al., 2003)
Analyte
Analyte
2
One Pulse Direct Observation of 15N in N-Sulfated Sugars
No proton decoupling (30mg non-enriched)
• Hyper polarization allows direct
observation (1 pulse with
enhancement of ~4000).
• For N-sulfated sugars proton exchange
at normal amide observation pH (~5)
is too rapid to allow indirect
detection. (C. Larive pointed out
solution)
• Without hyperpolarization acquisition
by signal averaging would have taken
more than a month of signal averaging
Current In Vivo Applications Utilize 13C Observation
and Pyruvate 13C Labeled at Carbonyl
Monitoring of pyruvate metabolism in TRAMP mouse (prostate model) with highgrade primary tumor – M. J. Albers et al., 2008, Cancer Research, 68:8607-8615
Metabolic 13C observation in HEK293 cells
8 mm NMR tube
Injection tube
Exit tube
100 million Cells in Agarose beads
Lactate
Pyruvate
9
A variety of other substrates can be used
*
*
*
*
Example: Fumaric acid to
malic acid conversion
indicates onset of acute
tubular necrosis of the
mouse kidney. Images
are 10 and 18 hrs after
folic acid induced
nephropathy. Left and
right images based on
signals of carboxyl
resonances of fumaric and
malic acid respectively.
Kevin M. Brindle, et al. PNAS, 109, 1374-1379, 2012
Polarization Storage Limits Experiment Time
Long relaxation times are desirable
T1 for N-D
B0 (Tesla)
T1 for C=O
B0 (Tesla)
Singlet storage in proton pairs can also be explored:
(-)/2
Experimental Effect of Deuterating Glutamine
Sensitivity can be improved by indirect detection
through protons – reintroduce these by H/D exchange
Indirect Detection of Polarized 15N by Amide Proton Exchange
Barb, Hekmatyar, Glushka, & Prestegard. (2011) J Mag Res 212:304-310.
Useful signal up to a minute
Sensitivity enhancement ~100,000 x direct 15N observe
Rapid injection and a high field
path helps preserve polarization.
CP from protons speeds build-up
Bowen S & Hilty C, (2010) Phys. Chem. Chem.
Phys, 12: 5766-5770 (thematic issue on DNP)
Bornet, A; Milani, J; Wang, S; Bodenhausen, G.,
(2012) CHIMIA 66: 734-740.
Jannin, S; Bornet, A; Melzi, R; Bodenhausen, G.
(2012) Chem. Phys. Lett. 549: 99-102.
Can one go to two dimensions? Improve
Resolution of Metabolites? – HSQC of Glucose
2nd Dimension Normally Collected a Point at a Time
t1
t1
FT
1
2
2
Ultra-Fast HSQC – 2D in 1s
Mishkovsky and Frydman, ChemPhysChem, 9:2340-2348 (2008)
Chirp
Chirp
RF
Mix
1
1
2
2
Gradients

Observe (echo-planar)
Z
t1
t1
t1
t1
t2
For each chemical shift, 1 = zG + ; 2 = -zG + 
1 = c 1; 2 = c 2; 1 + 2 = 2/c;  - 1 + 2 = n/ 
Therefore each element in tube has unique chemical
shift inversion – spatial encoding
t1
t1
t1
t1
Single Pulse 2D 1H-15N Spectrum
using D/H exchange enhanced DNP
H1
N15
Spatial encoding (N15): Donovan, K. & Frydman, L., JMR, 2012
Hyperpolarized 15N-Glutamine: Inverse 15N detection
t1 echo train
2D transform