Name: dissolution dynamic nuclear polarization instrumentation for real-time enzymatic reaction rate measurements by nmr
Uploaded: 2016-02-23T15:00:00
Description: Watch this Scientific Journal Video about Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR at JoVE.com

The authors thank Dr J. J. van der Klink for the assistance in the choice and assembly of the equipment, as well as Dr F. Kateb and Dr G. Bertho for useful discussions. A.C. was supported by the Swiss National Science Foundation (grant PPOOP2_157547). We acknowledge financing from Paris Sorbonne Cit&#233; (NMR@Com, DIM Analytics, Ville de Paris, the Fondation de la Recherche M&#233;dicale (FRM ING20130526708), and the Parteneriat Hubert Curien Brancusi 32662QK. Our team is part of Equipex programs Paris-en-R&#233;sonance and CACSICE.

NOTE: All data analysis was performed using commercial software.
1. Prepare the Polarizing Solution
<ol>
	<li>Prepare 2 ml of a 1.12 M 13C-labeled sodium pyruvate (Na+[CH3-CO-13COO]-, substrate) solution doped with 33 mM of TEMPOL radical (4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl, polarizing agent)4 in 2:1 D2O/d6-ethanol for 13C observations. CAUTION: Handling precautions must be taken due to the flammable nat...

<ol>
	<li>Overhauser, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polarization+of+Nuclei+in+Metals.">Polarization of Nuclei in Metals.</a> Phys. Rev. 92 (2), 411-415 (1953).</li><li>Abragam, A., Goldman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Principles+of+dynamic+nuclear+polarisation.">Principles of dynamic nuclear polarisation.</a> Rep. Prog. Phys. 41 (3), 395 (1978).</li><li>Wolber, J., Ellner, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+highly+polarized+nuclear+spins+in+solution+using+dynamic+nuclear+polarization.">Generating highly polarized nuclear spins in solution using dynamic nuclear polarization.</a> Nuc. Inst. Met. Phys. Res. Sec. A. 526 (1-2), 173-181 (2004).</li><li>Cheng, T., Capozzi, A., Takado, Y., Balzan, R., Comment, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Over+35%+liquid-state+13C+polarization+obtained+via+dissolution+dynamic+nuclear+polarization+at+7+T+and+1+K+using+ubiquitous+nitroxyl+radicals.">Over 35% liquid-state 13C polarization obtained via dissolution dynamic nuclear polarization at 7 T and 1 K using ubiquitous nitroxyl radicals.</a> PCCP. 15 (48), 20819-20822 (2013).</li><li>Ardenkjaer-Larsen, J. H., Fridlund, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increase+in+signal-to-noise+ratio+of+&gt;+10,000+times+in+liquid-state+NMR.">Increase in signal-to-noise ratio of &gt; 10,000 times in liquid-state NMR.</a> PNAS. 100 (18), 10158-10163 (2003).</li><li>Day, S. E., Kettunen, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+tumor+response+to+treatment+using+hyperpolarized+13C+magnetic+resonance+imaging+and+spectroscopy.">Detecting tumor response to treatment using hyperpolarized 13C magnetic resonance imaging and spectroscopy.</a> Nat. Med. 13 (11), 1382-1387 (2007).</li><li>Keshari, K. R., Wilson, D. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+[2-13C]-Fructose:+A+Hemiketal+DNP+Substrate+for+In+Vivo+Metabolic+Imaging.">Hyperpolarized [2-13C]-Fructose: A Hemiketal DNP Substrate for In Vivo Metabolic Imaging.</a> JACS. 131 (48), 17591-17596 (2009).</li><li>Zeng, H., Lee, Y., Hilty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Rate+Determination+by+Dynamic+Nuclear+Polarization+Enhanced+NMR+of+a+Diels&#8722;Alder+Reaction.">Quantitative Rate Determination by Dynamic Nuclear Polarization Enhanced NMR of a Diels&#8722;Alder Reaction.</a> An. Chem. 82 (21), 8897-8902 (2010).</li><li>Harrison, C., Yang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+kinetic+models+for+analysis+of+pyruvate-to-lactate+exchange+by+hyperpolarized+13C+NMR.">Comparison of kinetic models for analysis of pyruvate-to-lactate exchange by hyperpolarized 13C NMR.</a> NMR in Biom. 25 (11), 1286-1294 (2012).</li><li>Allouche-Arnon, H., Gamliel, A., Sosna, J., Gomori, J. M., Katz-Brull, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+visualization+of+betaine+aldehyde+synthesis+and+oxidation+using+hyperpolarized+magnetic+resonance+spectroscopy.">In vitro visualization of betaine aldehyde synthesis and oxidation using hyperpolarized magnetic resonance spectroscopy.</a> Chem. Comm. 49 (63), 7076-7078 (2013).</li><li>Lerche, M. H., Meier, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+dynamic+nuclear+polarization-NMR+on+blood+plasma+for+assays+of+drug+metabolism.">Quantitative dynamic nuclear polarization-NMR on blood plasma for assays of drug metabolism.</a> NMR in Biom. 24 (1), 96-103 (2011).</li><li>Nelson, S. J., Kurhanewicz, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+Imaging+of+Patients+with+Prostate+Cancer+Using+Hyperpolarized+[1-13C]Pyruvate.">Metabolic Imaging of Patients with Prostate Cancer Using Hyperpolarized [1-13C]Pyruvate.</a> Sci. Trans. Med. 5 (198), 198ra108 (2013).</li><li>Kurhanewicz, J., Vigneron, D. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+Cancer+Metabolism+by+Imaging+Hyperpolarized+Nuclei:+Prospects+for+Translation+to+Clinical+Research.">Analysis of Cancer Metabolism by Imaging Hyperpolarized Nuclei: Prospects for Translation to Clinical Research.</a> Neoplasia. 13 (2), 81-97 (2011).</li><li>Comment, A., Merritt, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperpolarized+Magnetic+Resonance+as+a+Sensitive+Detector+of+Metabolic+Function.">Hyperpolarized Magnetic Resonance as a Sensitive Detector of Metabolic Function.</a> Biochem. 53 (47), 7333-7357 (2014).</li><li>Carravetta, M., Johannessen, O. G., Levitt, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+the+T-1+limit:+Singlet+nuclear+spin+states+in+low+magnetic+fields.">Beyond the T-1 limit: Singlet nuclear spin states in low magnetic fields.</a> PRL. 92 (15), 153003 (2004).</li><li>Carravetta, M., Levitt, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory+of+long-lived+nuclear+spin+states+in+solution+nuclear+magnetic+resonance.+I.+Singlet+states+in+low+magnetic+field.">Theory of long-lived nuclear spin states in solution nuclear magnetic resonance. I. Singlet states in low magnetic field.</a> J. Chem. Phys. 122 (21), 214505 (2005).</li><li>Vasos, P. R., Comment, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+states+to+sustain+hyperpolarized+magnetization.">Long-lived states to sustain hyperpolarized magnetization.</a> PNAS. 106 (44), 18469-18473 (2009).</li><li>Claytor, K., Theis, T., Feng, Y., Warren, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+long-lived+13C2+state+lifetimes+at+natural+abundance.">Measuring long-lived 13C2 state lifetimes at natural abundance.</a> JMR. 239, 81-86 (2014).</li><li>Pileio, G., Carravetta, M., Hughes, E., Levitt, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-lived+nuclear+singlet+state+of+N-15-nitrous+oxide+in+solution.">The long-lived nuclear singlet state of N-15-nitrous oxide in solution.</a> JACS. 130 (38), 12582-12583 (2008).</li><li>Stevanato, G., Hill-Cousins, J. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Nuclear+Singlet+Lifetime+of+More+than+One+Hour+in+Room-Temperature+Solution.">A Nuclear Singlet Lifetime of More than One Hour in Room-Temperature Solution.</a> Ange. Chem. Int. Ed. 54 (12), 3740-3743 (2015).</li><li>Ghosh, R. K., Kadlecek, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurements+of+the+Persistent+Singlet+State+of+N(2)O+in+Blood+and+Other+Solvents-Potential+as+a+Magnetic+Tracer.">Measurements of the Persistent Singlet State of N(2)O in Blood and Other Solvents-Potential as a Magnetic Tracer.</a> MRM. 66 (4), 1177-1180 (2011).</li><li>Harris, T., Eliyahu, G., Frydman, L., Degani, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetics+of+hyperpolarized+13C1-pyruvate+transport+and+metabolism+in+living+human+breast+cancer+cells.">Kinetics of hyperpolarized 13C1-pyruvate transport and metabolism in living human breast cancer cells.</a> PNAS. 106 (43), 18131-18136 (2009).</li><li>Comment, A., van den Brandt, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+a+DNP+prepolarizer+coupled+to+a+rodent+MRI+scanner.">Design and performance of a DNP prepolarizer coupled to a rodent MRI scanner.</a> Conc. Mag. Res. B. 31 (4), 255-269 (2007).</li><li>Balzan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods for Molecular Magnetic Resonance Imaging and Magnetic Resonance Spectroscopy using Hyperpolarized Nuclei. 5966, 1-140 (2013).</li><li>Bornet, A., Melzi, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boosting+Dissolution+Dynamic+Nuclear+Polarization+by+Cross+Polarization.">Boosting Dissolution Dynamic Nuclear Polarization by Cross Polarization.</a> JPC Letters. 4 (1), 111-114 (2013).</li><li>Bowen, S., Hilty, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+injection+for+hyperpolarized+NMR+spectroscopy.">Rapid sample injection for hyperpolarized NMR spectroscopy.</a> PCCP. 12 (22), 5766-5770 (2010).</li><li>Cavadini, S., Vasos, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Singlet+states+open+the+way+to+longer+time-scales+in+the+measurement+of+diffusion+by+NMR+spectroscopy.">Singlet states open the way to longer time-scales in the measurement of diffusion by NMR spectroscopy.</a> Conc. Mag. Res. A. 32 (1), 68-78 (2008).</li><li>Ahuja, P., Sarkar, R., Vasos, P. R., Bodenhausen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-lived+States+in+Multiple-Spin+Systems.">Long-lived States in Multiple-Spin Systems.</a> Chem. Phys. Chem. 10 (13), 2217-2220 (2009).</li><li>Ahuja, P., Sarkar, R., Jannin, S., Vasos, P. R., Bodenhausen, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+hyperpolarisation+preserved+in+long-lived+states.">Proton hyperpolarisation preserved in long-lived states.</a> Chem. Comm. 46 (43), 8192-8194 (2010).</li><li>Sarkar, R., Comment, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proton+NMR+of+15N-Choline+Metabolites+Enhanced+by+Dynamic+Nuclear+Polarization.">Proton NMR of 15N-Choline Metabolites Enhanced by Dynamic Nuclear Polarization.</a> JACS. 131 (44), 16014-16015 (2009).</li></ol>

The critical points of the dissolution DNP NMR experiment are: (i) the level of polarization attained for the substrate, which determines the lowest product concentration necessary for experiments as well as the number of signal acquisitions that can be performed and (ii) the lifetimes of magnetization, compared to the duration of the transfer between the polarization and the detection sites and to the rate of substrate transformation. The injection system of the dissolution DNP setup herein described allows for sample t...

Dynamic nuclear polarization (DNP),1,2 a technique designed to enhance the nuclear spin polarization, i.e., the imbalance between &#39;up&#39; and &#39;down&#39; spin populations (P = [N&#8593; - N&#8595;] / [N&#8593; + N&#8595;]), was first introduced in the 1950&#39;s. Nuclear spins such as 13C can be polarized up to P = 10-1 in favorable conditions, typically at a temperature on the order of 1 K and in a magnetic field of 3.357 T.3,4 A breakthrough for biological applications came in the early 2000&#39;s with the development of dissolution DNP which consists in dissolving frozen polarized samples in superheated water while retaining the high nuclear polarization level obtained at low temperature.5 The liquid-state NMR signal is enhanced by a factor 103-104 as compared to common thermally-polarized RT NMR conditions. Dissolution DNP therefore provides a way to non-invasively measure biochemical reaction rates in situ in realtime, allowing monitoring dynamics by NMR with a temporal resolution of 1 sec or less.6-10 It also became possible to detect analytes in very low concentrations.11
Among the non-invasive molecular imaging modalities, hyperpolarized NMR is the only technique that allows simultaneously measuring a substrate and its metabolic products in real-time. Dissolution DNP was received with enthusiasm in various scientific areas ranging from in vitro NMR to clinical MRI12 and the most promising applications are related to the in situ monitoring of metabolism.13,14 The main limitation of dissolution DNP is that, after a time on the order of five times the longitudinal relaxation time constant T1, the enhanced polarization is lost. It is therefore necessary to use molecules bearing nuclear spins exhibiting relatively long T1. To extend the time-span of the polarization enhancement, slowly-relaxing nuclear spin states, known as long-lived states (LLS), may be used.15-17 LLS are insensitive to the intra-pair dipole-dipole interaction, so their characteristic relaxation time constant, TLLS, can be much longer than T1.18 A magnetization lifetime of tens of minutes and up to 1 hr could therefore be obtained,19,20 and LLS have been proposed for both magnetic resonance spectroscopy (MRS) and MRI.21
The main points that need to be carefully optimized for studying enzymatic reaction rates by hyperpolarized NMR are: (i) maximize the solid-state polarization and (ii) minimize the polarization loss during the transfer of the hyperpolarized solution from the polarizer to the NMR spectrometer. This article describes the adaptation of a custom-made dissolution DNP apparatus and injection system to study enzymatic reactions. The characteristics and performance of the setup will be demonstrated with the well-known and widely-used hyperpolarized substrate [1-13C]pyruvate. The main reasons for this choice are, first, its naturally long 13C longitudinal relaxation time (T1 &#62;50 sec at high magnetic fields and temperatures above 293 K) which allows monitoring reactions during several minutes, and, second, its central role in cancer metabolism.13,14 Using dissolution DNP NMR and a custom-developed injection system, the oxidation of pyruvate catalyzed by lactate dehydrogenase (LDH) can be monitored in presence of an initial pool of unlabelled lactate9,22 or with no unlabelled lactate added, as shown here. It has been shown that the [1-13C]lactate signal measured in vivo (including in cells) following the injection of hyperpolarized [1-13C]pyruvate is mainly due to a fast label exchange between pyruvate and lactate rather than to lactate production.6
We herein present the real-time production of [1-13C]lactate from hyperpolarized [1-13C]pyruvate injected into a NMR tube containing LDH but initially no lactate.
System description 
There are two main parts in a dissolution DNP setup (Figure 1): the DNP polarizer and the NMR spectrometer. The main element of the DNP polarizer is a cryostat to cooling the sample to around 1 K in a pumped helium bath. The cryostat is inserted in a 3.35 T superconductive magnet and has a geometry that guarantees to have the polarizing sample at the isocenter of the magnet (Figure 1). Inside the cryostat, the sample (a) is surrounded by a NMR coil (b), to measure the polarization buildup, contained in an overmoded microwave cavity (c). The whole sample is kept at low temperature in a pumped helium bath (d) and irradiated with microwaves through the waveguide. The whole system is managed by custom-made software (Figure 2D).
The hardware and cryogenic equipment needed to perform DNP and the subsequent dissolution are still a technological challenge. A new DNP cryostat23,24 was developed and tested to determine its cryogenic performances and then optimized for fast cool-down, helium hold-time and overall minimal helium consumption during operation.
The cryostat consists of two parts. The first part of the cryostat is the insulation dewar (Figure 2A) that can be roughly separated in top part (a) the tail, or sample space (b), and the outer vacuum chamber (OVC) kept under high vacuum and housing the radiation screens (c). The second part of the cryostat is the main insert (Figure 2B), placed into the insulation dewar, where all the flow regulations are managed. The liquid helium coming from the external storage dewar through the transfer line (a), is in the first stage condensed in the separator (b), an intermediate chamber used both to keep the top part of the cryostat cold and to remove the helium evaporated during the transfer. The separator pressure is lowered by pumping through a capillary (c) wrapped around the top part of the cryostat; the flow of cold helium in this capillary is used to cool down the baffles (d) and the radiation screens in the insulation dewar (OVC). The sample is placed and polarized in the sample space. The sample space is connected to the separator through another capillary (e), wrapped around the tail of the main cryostat insert. This capillary can be opened or closed through a needle valve manually operated from outside.
To achieve the low temperature used during the DNP process, liquid helium needs to be collected in the cryostat sample space and its pressure lowered to the mbar range. The operations needed for cryostat operation are performed through a rather complex pumping system with three sets of pumps, monitored and operated in different points with electronic and electro-mechanic instruments (Figure 2C). The cryostat OVC needs to be pumped to high vacuum by the first pumping system. This system is composed of a turbo-molecular pump backed up by a rotary pump (a). The liquid helium is transferred from the storage dewar (b) through the cryostat transfer line inlet to the cryostat separator. The separator has an outlet connected to the second pumping set. This set is composed of a 35 m3/hr membrane pump (c). This line allows removing the helium gas boiled during the transfer from the dewar and during separator cooling. The liquid helium collected in the separator can then be transferred to the sample space through the capillary tubes described above. To transfer liquid helium from the separator to the sample space and subsequently to lower sample space pressure to mbar range, a third pumping system composed of a 250 m3/hr Roots pump backed up by a 65 m3/hr rotary pump (d) is connected to the cryostat through a manual butterfly valve (e).
All the vacuum system operations are controlled and regulated by an electropneumatic custom-made device (f). This device controls vacuum line connections between the cryostat separator (g) and sample space (h) outlets, the second/third pumping systems (c, d), a compressed helium bottle (i) and the outside. Communication between (f) and the outside passes through a one-way valve (j). The electro-pneumatic device (f) as well as all the system parameters and the dissolution hardware are controlled and operated by a custom-made electronic device interfaced USB with a common PC. Finally all the system, through the electronic device, is managed by custom-made standalone software (Figure 2D) where relevant operations are launched through an interface using software buttons.
To manage the sample and measure NMR signal build-up in the solid state a series of inserts are used (Figure 3A). To prepare the cryostat for polarization, place the main sample insert (a), into the cryostat. The main sample insert is provided with an NMR coil (b) placed inside an overmoded gold-plated microwave cavity. Pre-freeze the substrate containing solution to be polarized (polarizing solution) at liquid nitrogen temperature in a suitable sample container and place it at the end bottom of the fiberglass sample holder (c). Slide the sample holder into the main sample insert to reach the magnet isocenter. Insert the gold-plated waveguide (d) in the sample holder. The waveguide allows the microwave generated from an external microwave source to travel with minimal losses to the sample.
The custom-made software for cryostat management handles automatically, upon clicking the corresponding interface button, different operations like cooldown (the cryostat temperature is lowered close to liquid helium temperature), filling (the cryostat is filled with liquid helium to a pre-determined level), an additional step of cooling to T&#160;&#8776; 1 K (the liquid helium bath is pumped to achieve the lowest temperature possible), pressurization (the cryostat is pressurized slightly above room pressure at P = 10-30 mbar to allow cryostat opening without risks of contamination of the cryostat by air) and dissolution (automatic procedure to dissolve the DNP sample and transfer the resulting hyperpolarized solution to the measurement site, i.e., the NMR spectrometer).
The polarization is performed irradiating the sample with microwaves at 94 GHz (in a polarizing field B0 = 3.35 T). A sample is considered completely polarized after 3 TDNP, where TDNP is the polarization buildup time. TDNP is of the same order of magnitude as the longitudinal relaxation time of the target nuclei in solid state at the given field and temperature. In all our experiments the sample was polarized for more than 5 TDNP.
At the end of the polarization time, the sample has to be dissolved in a RT solution in order to be used for measurement of enzymatic activity. During the dissolution process, 5 ml of superheated D2O from the boiler of the dissolution insert (Figure 3B) are pushed by compressed helium gas (P = 6-8 bar) to reach the DNP-enhanced sample and dissolve it. The resulting hyperpolarized solution is pushed out the dissolution insert by the compressed helium gas, through the dissolution insert outlet (Figure 3C-b), a 2 mm inner diameter Teflon transfer tube. The time needed for the dissolution process is 300 msec.23 The time needed for the sample transfer from the DNP polarizer to the NMR spectrometer site is about 3 sec.
The dissolution process is performed using a dissolution insert (Figure 3B). The dissolution insert is composed of an electronic-pneumatic assembly (a), a carbon fiber stick (b) containing connection tubes between the boiler in the pneumatic assembly and the sample container locker (c), which allows leak-tight coupling with the sample container, and back out to the outlet. The electro-pneumatic assembly (Figure 3C) is used to produce and drive superheated D2O through the carbon fiber stick to the sample container and then to extract the hyperpolarized solution from the cryostat. The electro-pneumatic assembly is composed of pneumatic valves (a) that control the connections between the compressed helium (P = 6-8 bar) line (b), the boiler (c) where the D2O is injected through the valve (d), and the outlet (e) through the carbon fiber stick (f). The system is completed by a pressure G, a thermometer and a heating resistive wire in the boiler (c), a trigger (h) and a connection box (i) used to interface the system with the electronic management device.
The DNP cryostat and the NMR spectrometer are connected by a transfer line, i.e., a PTFE tube of 2 mm inner diameter inside which the hyperpolarized solution is pushed by pressurized helium (P = 6-8 bar) when dissolution is triggered.
The dissolution sequence is composed of the following operations: in the first 300 msec, superheated D2O is pushed to the sample container in order to melt and dissolve the hyperpolarized frozen solution. Afterwards, the hyperpolarized solution is extracted from the cryostat by mean of pressurized (P = 6-8 bar) helium gas and pushed through the 2 mm inner diameter PTFE tube (Figure 3C-e) to the measurement site where the injection is performed with either of the procedures described in Step 6.2.1 or Step 6.2.2.
The second component of the dissolution DNP NMR setup is the NMR spectrometer. In the setup described herein, the NMR spectrometer operates at a field B0 = 11.7 Tesla. A 5 mm NMR probe is used to measure the hyperpolarized signal after the dissolution. The NMR spectrometer is operated through the NMR console, used for both solid-state and liquid-state NMR measurements, and the firm-provided software XWinNMR. A typical measurement is composed of a low flip angle hard pulse (either calibrated, for liquidstate or un-calibrated, for solid-state measurements) followed by signal acquisitions.
Measurements of the solid state thermal polarization signal and DNP-derived signal build-up are performed using the custom-made 13C coil at the site of the DNP polarizer (Figure 3Ab) coupled to the NMR spectrometer. In this particular situation the NMR spectrometer does not perform signal locking. When solid-state measurements are carried out, to avoid significant perturbations to the polarization, the time delay between acquisitions should be long enough, roughly longer than 0.5 TDNP.
The solid-state enhancement is defined as <img alt="Equation4" src="/files/ftp_upload/53548/53548equation4.jpg" width="100" /> where <img alt="Equation5" src="/files/ftp_upload/53548/53548equation5.jpg" width="31" /> is the hyperpolarized signal (obtained in Step 3.3) and <img alt="Equation6" src="/files/ftp_upload/53548/53548equation6.jpg" width="23" /> is the solid state signal (obtained at thermal equilibrium at pumped liquid helium temperature in Step 3.2) (Figure 4A). This parameter defines the maximal polarization available for NMR experiments, prior to unavoidable losses during the transfer of the hyperpolarized solution. The measurement is performed with a simple pulse-acquire sequence using an un-calibrated low flip angle pulse. Pulse calibration is commonly skipped for solidstate measurements.
An analogous procedure can be used to determine the hyperpolarized signal enhancement in the liquid-state. In this case, the sample placed in the spectrometer tube before the injection (Step 6.2) is composed of 500 &#181;l of D2O. After dissolution and injection, there are two important parameters to monitor. The first is the hyperpolarized enhancement at the NMR spectrometer site, <img alt="Equation7" src="/files/ftp_upload/53548/53548equation7.jpg" width="95" /> (Figure 4B), where <img alt="Equation8" src="/files/ftp_upload/53548/53548equation8.jpg" width="30" /> is the signal just after the injection of the hyperpolarized solution (obtained in Step 7.1) and <img alt="Equation9" src="/files/ftp_upload/53548/53548equation9.jpg" width="20" /> is the thermal polarization signal (obtained in Step 7.2). The second is the longitudinal relaxation time, T1 (Figure 4B, inset), associated with the substrate and each metabolic product (obtained by exponential fitting signals obtained in Step 7.1). These two parameters define the minimum substrate concentration necessary to obtain a sufficient signal-to-noise ratio (SNR) and the available time window for the measurement of the metabolic transformations. The ratio between solid-state polarization <img alt="Equation10" src="/files/ftp_upload/53548/53548equation10.jpg" width="30" /> and liquidstate polarization <img alt="Equation11" src="/files/ftp_upload/53548/53548equation11.jpg" width="29" /> gives an estimate of the polarization losses due to relaxation during the hyperpolarized solution transfer. A value <img alt="Equation12" src="/files/ftp_upload/53548/53548equation12.jpg" width="80" /> should be observed in absence of relaxation losses.

<table border="0" cellpadding="0" cellspacing="0" width="789">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNP polarizer</td>
			<td style="width:287px;">Vanderklink s.a.r.l (Switzerland)</td>
			<td style="width:119px;">///</td>
			<td style="width:167px;">Cryostat and electronic equipment for sample polarization</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vacuum system components</td>
			<td style="width:287px;">Edwards vacuum (France)</td>
			<td style="width:119px;">Various</td>
			<td>
			- turbomolecular pumping setup
			- membrane pumping setup
			- high capacity roots pumping system
			- vacuum fittings and components
			</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DNP 3.35T Magnet</td>
			<td style="width:287px;">Bruker (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">500MHz NMR Spectrometer</td>
			<td style="width:287px;">Bruker (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Origin 8.0</td>
			<td style="width:287px;">OriginLab (US)</td>
			<td style="width:119px;"></td>
			<td>Data analysis software</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chemicals</td>
			<td style="width:287px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SODIUM PYRUVATE-1-13C, 99 ATOM % 13C</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">490709</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ETHANOL-D6, ANHYDROUS, 99.5 ATOM % D</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">186414</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#160;4-Hydroxy-TEMPO 97%</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">176141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Deuterium oxide</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td align="right" style="width:119px;">151882</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">reduced nicotinamide adenine dinucleotide (NADH)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethylene-diaminetetraacetic acid (EDTA)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dithiothreitol (DTT)</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">phosphate buffer, pH&#160;=&#160;7.0</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LDH enzyme in&#160;</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;">L-2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">bovine serum albumin, BSA</td>
			<td style="width:287px;">Sigma Aldrich (France)</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

dissolution dynamic nuclear polarization instrumentation for real-time enzymatic reaction rate measurements by nmr

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of metabolic transformations. Hyperpolarization via dissolution DNP circumvents part of the sensitivity issues thanks to the large out-of-equilibrium nuclear magnetization stemming from the electron-to-nucleus spin polarization transfer. The high NMR signal obtained can be used to monitor chemical reactions in real time. The downside of hyperpolarized NMR resides in the limited time window available for signal acquisition, which is usually on the order of the nuclear spin longitudinal relaxation time constant, T1, or, in favorable cases, on the order of the relaxation time constant associated with the singlet-state of coupled nuclei, TLLS. Cellular uptake of endogenous molecules and metabolic rates can provide essential information on tumor development and drug response. Numerous previous hyperpolarized NMR studies have demonstrated the relevancy of pyruvate as a metabolic substrate for monitoring enzymatic activity in vivo. This work provides a detailed description of the experimental setup and methods required for the study of enzymatic reactions, in particular the pyruvate-to-lactate conversion rate in presence of lactate dehydrogenase (LDH), by hyperpolarized NMR.

NMR signal gains using dissolution DNP 
The DNP effect consists in the transfer of the high polarization of unpaired electron spins, typically from stable radical molecules, to NMR-active nuclei, under microwave irradiation of the sample. The most often-used free radicals are TAM(OXO63) and TEMPOL.4 Polarization procedures using TEMPOL may be optimized by &#39;cross-polarization&#39;.25
Optimizing the ...

Watch this Scientific Journal Video about Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR at JoVE.com

Dissolution Dynamic Nuclear Polarization Instrumentation for Real-time Enzymatic Reaction Rate Measurements by NMR

The sensitivity enhancement provided by dissolution dynamic nuclear polarization (DNP) enables following metabolic processes in real time by NMR and MRI. The characteristics and performances of a dedicated dissolution DNP setup designed for study enzymatic reactions are discussed.

The main limitation of NMR-based investigations is low sensitivity. This prompts for long acquisition times, thus preventing real-time NMR measurements of ...

The overall goal of this experiment is to follow enzymatic processes in real time by dynamic nuclear polarization enhanced nuclear magnetic resonance. Magnetic resonance in the liquid state enhanced by the dynamic nuclear polarization, DNP, can help answer key questions:how to determine, for instance, the rates of enzymatic transformation of a pair of molecules in cell or in vivo without having to alter this pair of molecules in any way. Installation of our dissolution DNP equipment in our laboratory and the arrival here of Dr.Riccardo Balzan, a PhD in Arnaud Comment Laboratory in Lausanne, is a big step forward for our research.DNP significantly increases the sensitivity of experiments we can perform on our 500 megahertz NMR spectrometers. This DNP equipment was the first one to be installed in France and I would like to mention that, in rendering this equipment functional, all the members of our NMR team were involved and I would like to mention in particular Gildas Bertho, our research engineer, and Fatiha Kateb, our maitre de conferences. The main advantage of this technique is that it allows direct, real-time quantification of the substrate and its endogenous derivates with a time resolution of seconds.For the polarizing solution, prepare two milliliters of a 1.12 molar, carbon 13 labeled sodium pyruvate solution doped with 33 millimolar of TEMPO radical in a two-to-one volume of deuterated water to deuterated ethanol for carbon 13 observations. Lower the dynamic nuclear polarization, or DNP, cryostat temperature through the cooldown procedure by clicking the cooldown button on the cryostat software interface. Collect liquid helium in the DNP cryostat through the filling procedure.Click the filling button on the cryostat software interface. Then place 300 microliters of the optimized polarizing solution in the sample container provided with the DNP polarizer. Freeze the sample container with the sample inside by gently immersing it in a liquid nitrogen bath.Eliminate the liquid nitrogen that may be present in the sample container after the freezing procedure by extracting the sample container from the liquid nitrogen bath and rotating it upside down. Next, insert the sample into the cryostat by first inserting the sample container in the sample holder. Then place the sample holder in the main cryostat insert before inserting the microwave's wave guide into the sample holder.Lower the cryostat temperature by starting the one Kelvin cooling procedure. Click the one Kelvin cooling button on the cryostat software interface. Turn the microwave source on and irradiate the sample by clicking the microwaves on button on the cryostat software interface.Disconnect the coaxial cable of the NMR console detection channel from the spectrometer coil by rotating the connector anti-clockwise by 90 degrees and pulling it. Plug in the coaxial cable of the detection channel to the coaxial connector of the cryostat NMR coil. Then insert the male connector of the NMR console cable in the female connector of the cryostat NMR coil, paying attention to the orientation pin.Push firmly and rotate the connector clockwise by 90 degrees. Measure the NMR thermal signal in the solid state at the cryostat site using a simple pulse-acquire sequence with uncalibrated, low flip angle pulses repeated at an interval of t equals five minutes. After setting up the measurement, write zg in the command line of the software and press enter on the keyboard.After initiating the polarization procedure, measure the DNP enhanced NMR signal in the solid state with the same procedure and conditions used for the thermal signal measurement. Disconnect the NMR console detection channel coaxial cable from the cryostat NMR coil by rotating the connector anti-clockwise by 90 degrees and pulling it. Then plug the coaxial cable of the detection channel to the coaxial connector of the spectrometer coil.Insert the male connector of the NMR console cable in the female connector of the spectrometer coil, paying attention to the orientation pin. Push firmly and rotate the connector clockwise by 90 degrees. Freshly prepare a rabbit muscle LDH solution at one unit per milliliter in reaction buffer with 20 micrograms per milliliter bovine serum albumin.Next mix 478 microliters of reaction buffer, 2 microliters of LDH solution, and 20 microliters of deuterated water to allow for spectrometer field stabilization, or locking. Then place the 500 microliter solution volume into a 5 millimeter NMR tube. Connect the transfer tube to an automated, custom-made injection device that separates the hyperpolarized solution from the helium gas used for transfer.The device automatically delivers 500 microliters of hyperpolarized solution into the sample tube. Before dissolution, place the five millimeter NMR tube containing 500 microliters of sample in the isocenter of the 11.74 tesla NMR spectrometer. Insert 5 milliliters of deuterated water in the dissolution insert boiler.Pressurize and heat the deuterated water by means of the resistive wire until a temperature of approximately 180 to 200 degrees Celsius is reached by clicking the prepare heater button on the cryostat software interface. Click the SS Operations button on the cryostat software interface. Then remove the microwave guide from the dissolution insert.Slide the dissolution insert down into the sample holder. The dissolution insert has to reach the bottom of the sample holder and must be pushed firmly to make a leak-tight connection with the sample container to avoid deuterated water leaking into the cryostat. Press the hardware trigger to begin the dissolution sequence, a predetermined timed sequence of pneumatic valves operations.Just after the dissolution, the injection device mixes 500 microliters of hyperpolarlized solution with the sample placed in the NMR spectrometer. After the dissolution, measure the NMR hyperpolarized signal from the sample in the spectrometer by a series of 10 degree, flip angle pulse-acquire sequences spaced by 1.5 seconds to follow the progression of magnetization of the substrate and the product. After setting up the measurement, write zg in the command line of the software and press enter.Once the magnetization is fully relaxed, measure the NMR thermal signal by a series of 90 degree flip angle pulse-acquire sequences spaced by about three minutes, corresponding to about 3T1. After setting up the measurement, write zg in the command line of the software the press the enter key. Here the time course of spectra obtained from repeated 10 degree flip angle pulses show the sensitivity and time resolution of the technique.Following peak identification of the pyruvate substrate and lactate product, quantification is performed through signal integration of the metabolites. Enzymatic activity is determined from the product signal time course by fitting with a linear model. To validate the measurement, the same quantity is evaluated by the initial reaction rate, from the ratio of signals of product and substrate.Once mastered, a complete measurement of enzymatic activity can be performed in about two hours if it is performed properly. After watching this video, you should have an understanding of how to produce a highly polarized substrate for NMR, inject it into a solution containing an enzyme, and follow enzymatic activity using DNP enhanced NMR. When attempting this procedure, it is important to carefully prepare the enzymatic solution with fresh enzyme and cosubstrates, and to avoid any air bubble in the sample tube.With the development of DNP enhanced magnetic resonance in the liquid state, researchers can now explore substrate uptake and its enzymatic transformation inside various types of cells by measuring the rate of product formation. Don't forget that working with cryogenic equipment and high magnetic fields can be extremely hazardous. Precautions, such as wearing personal protections against cryogens and verifying the absence of magnetic objects should always be taken while performing these measurements.Unlike classical NMR, dissolution DNP based NMR is limited to an experimental time window of minutes at most, depending on the substrate and the current state of life. This stands in the way of going beyond one D spectroscopy. Dissolution DNP NMR needs development to bring it to the versatility level of classical multi-dimensional NMR.

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Unraveling Entropic Rate Acceleration Induced by Solvent Dynamics in Membrane Enzymes

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually neglected. The study of water dynamics in enzymes and the associated thermodynamical consequences is highly complex and has involved computer simulations, nuclear magnetic resonance (NMR) experiments, and calorimetry. Water tunnels that connect the active site with the surrounding solvent are key to solvent displacement and dynamics. The protocol herein allows for the engineering of these motifs for water transport, which affects specificity, activity and thermodynamics. By providing a biophysical framework founded on theory and experiments, the method presented herein can be used by researchers without previous expertise in computer modeling or biophysical chemistry. The method will advance our understanding of enzyme catalysis on the molecular level by measuring the enthalpic and entropic changes associated with catalysis by enzyme variants with obstructed water tunnels. The protocol can be used for the study of membrane-bound enzymes and other complex systems. This will enhance our understanding of the importance of solvent reorganization in catalysis as well as provide new catalytic strategies in protein design and engineering.

Channels for the transportation of water molecules in enzymes influence active site solvation and catalysis. Herein we present a protocol for the engineering of these additional catalytic motifs based on in silico computer modeling and experiments. This will enhance our understanding of the influence of solvent dynamics on enzyme catalysis.

Enzyme catalysis evolved in an aqueous environment. The influence of solvent dynamics on catalysis is, however, currently poorly understood and usually ...

Light-driven Enzymatic Decarboxylation

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. Recycling of the electron donors NADH or NADPH requires the use of additional enzymes and sacrificial substrates. Interestingly, several oxidoreductases accept hydrogen peroxide as electron donor. While being inexpensive, this reagent often reduces the stability of enzymes. A solution to this problem is the in situ generation of the cofactor. The continuous supply of the cofactor at low concentration drives the reaction without impairing enzyme stability. This paper demonstrates a method for the light-catalyzed in situ generation of hydrogen peroxide with the example of the heme-dependent fatty acid decarboxylase OleTJE. The fatty acid decarboxylase OleTJE was discovered due to its unique ability to produce long-chain 1-alkenes from fatty acids, a hitherto unknown enzymatic reaction. 1-alkenes are widely used additives for plasticizers and lubricants. OleTJE has been shown to accept electrons from hydrogen peroxide for the oxidative decarboxylation. While addition of hydrogen peroxide damages the enzyme and results in low yields, in situ generation of the cofactor circumvents this problem. The photobiocatalytic system shows clear advantages regarding enzyme activity and yield, resulting in a simple and efficient system for fatty acid decarboxylation.

We describe a protocol for the light-catalyzed generation of hydrogen peroxide &#8212; a cofactor for oxidative transformations.

Oxidoreductases belong to the most-applied industrial enzymes. Nevertheless, they need external electrons whose supply is often costly and challenging. ...

Cooling Rate Dependent Ellipsometry Measurements to Determine the Dynamics of Thin Glassy Films

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements are simple high-throughput characterization experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glassy states for a variety of glassy materials. This technique allows for these parameters to be measured in a single experiment, while other methods must combine a variety of different techniques to investigate all of these properties. Measurements of dynamics close to Tg are particularly challenging. The advantage of cooling rate dependent Tg measurements over other methods which directly probe bulk and surface relaxation dynamics is that they are relatively quick and simple experiments, which do not utilize fluorophores or other complicated experimental techniques. Furthermore, this technique probes the average dynamics of technologically relevant thin films in temperature and relaxation time (&#964;&#945;) regimes relevant to the glass transition (&#964;&#945; &#62; 100 sec). The limitation to using ellipsometry for cooling rate dependent Tg experiments is that it cannot probe relaxation times relevant to measurements of viscosity (&#964;&#945; &#60;&#60; 1 sec). Other cooling rate dependent Tg measurement techniques, however, can extend the CR-Tg method to faster relaxation times. Furthermore, this technique can be used for any glassy system so long as the integrity of the film remains throughout the experiment.

Here, we present a protocol for cooling rate dependent ellipsometry experiments, which can determine the glass transition temperature (Tg), average dynamics, fragility and the expansion coefficient of the super-cooled liquid and glass for a variety of glassy materials.

This report aims to fully describe the experimental technique of using ellipsometry for cooling rate dependent Tg (CR-Tg) experiments. These measurements ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Measurement of Particle Size Distribution in Turbid Solutions by Dynamic Light Scattering Microscopy

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a technique used to measure the size distribution of polymer solutions or colloidal particles. Although this technique is widely used for the assessment of polymer solutions, it is difficult to measure the particle size in concentrated solutions due to the multiple scattering effect or strong light absorption. Therefore, the concentrated solutions should be diluted before measurement. Implementation of the confocal optical component in a dynamic light scattering microscope1 helps to overcome this barrier. Using such a microscopic system, both transparent and turbid systems can be analyzed under the same experimental setup without a dilution. As a representative example, a size distribution measurement of a temperature-responsive polymer solution was performed. The sizes of the polymer chains in an aqueous solution were several tens of nanometers at a temperature below the lower critical solution temperature (LCST). In contrast, the sizes increased to more than 1.0 &#181;m when above the LCST. This result is consistent with the observation that the solution turned turbid above the LCST.

A protocol for the direct measurement of particle size distribution in concentrated solutions using dynamic light scattering microscopy is presented.

A protocol for measuring polydispersity of concentrated polymer solutions using dynamic light scattering is described. Dynamic light scattering is a ...

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

We describe a method to produce U2O5 films in situ using the Labstation, a modular machine developed at JRC Karlsruhe. The Labstation, an essential part of the Properties of Actinides under Extreme Conditions laboratory (PAMEC), allows the preparation of films and studies of sample surfaces using surface analytical techniques such as X-ray and ultra-violet photoemission spectroscopy (XPS and UPS, respectively). All studies are made in situ, and the films, transferred under ultra-high vacuum from their preparation to an analyses chamber, are never in contact with the atmosphere. Initially, a film of UO2 is prepared by direct current (DC) sputter deposition on a gold (Au) foil then oxidized by atomic oxygen to produce a UO3 film. This latter is then reduced with atomic hydrogen to U2O5. Analyses are performed after each step involving oxidation and reduction, using high-resolution photoelectron spectroscopy to examine the oxidation state of uranium. Indeed, the oxidation and reduction times and corresponding temperature of the substrate during this process have severe effects on the resulting oxidation state of the uranium. Stopping the reduction of UO3 to U2O5 with single U(V) is quite challenging; first, uranium-oxygen systems exist in numerous intermediate phases. Second, differentiation of the oxidation states of uranium is mainly based on satellite peaks, whose intensity peaks are weak. Also, experimenters should be aware that X-ray spectroscopy (XPS) is a technique with an atomic sensitivity of 1% to 5%. Thus, it is important to obtain a complete picture of the uranium oxidation state with the entire spectra obtained on U4f, O1s, and the valence band (VB). Programs used in the Labstation include a linear transfer program developed by an outside company (see Table of Materials) as well as data acquisition and sputter source programs, both developed in-house.

U2O5 Film Preparation via UO2 Deposition by Direct Current Sputtering and Successive Oxidation and Reduction with Atomic Oxygen and Atomic Hydrogen

This protocol presents the preparation of U2O5 thin films obtained in situ under ultra-high vacuum. The process involves oxidation and reduction of UO2 films with atomic oxygen and atomic hydrogen, respectively.

We describe a method to produce U2O5 films in situ using the Labstation, a modular machine developed at JRC Karlsruhe. The Labstation, an essential part ...

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands for in vivo investigations. Within this protocol, a robust and reliable remote-controlled radiosynthesis of [11C]SNAP-7941, an antagonist to the melanin-concentrating hormone receptor 1, is described. The radiosynthesis starts with cyclotron produced [11C]CO2 that is subsequently further reacted via a gas-phase transition to [11C]CH3OTf. Then, this reactive intermediate is introduced to the precursor solution and forms the respective radiotracer. Chemical as well as the radiochemical purity are determined by means of RP-HPLC, routinely implemented in the radiopharmaceutical quality control process. Additionally, the molar activity is calculated as it is a necessity for the following real-time kinetic investigations. Furthermore, [11C]SNAP-7941 is applied to MDCKII-WT and MDCKII-hMDR1 cells for evaluating the impact of P-glycoprotein (P-gp) expression on cell accumulation. For this reason, the P-gp expressing cell line (MDCKII-hMDR1) is either used without or with blocking prior to experiments by means of the P-gp substrate (&#177;)-verapamil and the results are compared to the ones observed for the wildtype cells. The overall experimental approach demonstrates the importance of a precise time-management that is essential for every preclinical and clinical study using PET tracers radiolabelled with short-lived nuclides, such as carbon-11 (half-life: 20 min).

Technical Aspect of the Automated Synthesis and Real-Time Kinetic Evaluation of [11C]SNAP-7941

Here, we represent a protocol for the fully automated radiolabelling of [11C]SNAP-7941 and the analysis of the real-time kinetics of this PET-tracer on P-gp expressing and non-expressing cells.

Positron emission tomography (PET) is an essential molecular imaging technique providing insights into pathways and using specific targeted radioligands ...

Niobium Oxide Films Deposited by Reactive Sputtering: Effect of Oxygen Flow Rate

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition parameters such as gas flow rate that results in changes on composition and thus in the film required properties. In this report, reactive sputtering is used to deposit niobium oxide films. A niobium target is used as metal source and different oxygen flow rates to deposit niobium oxide films. The oxygen flow rate was changed from 3 to 10 sccm. The films deposited under low oxygen flow rates show higher electrical conductivity and provide better perovskite solar cells when used as electron transport layer.

Here, we present a protocol for niobium oxide films deposition by reactive sputtering with different oxygen flow rates for use as an electron transport layer in perovskite solar cells.

Reactive sputtering is a versatile technique used to form compact films with excellent homogeneity. In addition, it allows easy control over deposition ...