Cryogenic Probe Research Articles

Four-Way Data Coupled to Parallel Factor Model Applied to Environmental Analysis: Determination of 2,3,7,8-Tetrachloro-dibenzo-para-dioxin in Highly Contaminated Waters by Solid−Liquid Extraction Laser-Excited Time-Resolved Shpol'skii Spectroscopy

This article reports the first application of parallel factor analysis to high-order instrumental data generated from Shpol'skii matrixes at liquid helium temperature. Third-order data arrays-consisting of excitation modulated wavelength time matrixes-are collected with the aid of a cryogenic fiber-optic probe, a tunable dye laser, and a multichannel system for phosphorescence detection. The multidimensional data formats are applied to the analysis of 2,3,7,8-tetrachloro-dibenzo-para-dioxin in water samples. The experimental procedure is rapid and environmentally friendly. Complete sample analysis is accomplished in less than 15 min with only 100 muL of organic solvent (n-heptane). The feasibility to directly determine parts-per-trillion concentration levels of the target compound is demonstrated with heavily contaminated samples of unknown composition. The limits of detection, estimated from calibrations based on the univariate method and parallel factor analysis, are 0.060 and 0.092 ng.mL(-1), respectively; both referred to a 100 mL water sample.

Structure and Dynamics of Micelle-bound Human α-Synuclein

Misfolding of the protein alpha-synuclein (aS), which associates with presynaptic vesicles, has been implicated in the molecular chain of events leading to Parkinson's disease. Here, the structure and dynamics of micelle-bound aS are reported. Val3-Val37 and Lys45-Thr92 form curved alpha-helices, connected by a well ordered, extended linker in an unexpected anti-parallel arrangement, followed by another short extended region (Gly93-Lys97), overlapping the recently identified chaperone-mediated autophagy recognition motif and a highly mobile tail (Asp98-Ala140). Helix curvature is significantly less than predicted based on the native micelle shape, indicating a deformation of the micelle by aS. Structural and dynamic parameters show a reduced helical content for Ala30-Val37. A dynamic variation in interhelical distance on the microsecond timescale is complemented by enhanced sub-nanosecond timescale dynamics, particularly in the remarkably glycine-rich segments of the helices. These unusually rich dynamics may serve to mitigate the effect of aS binding on membrane fluidity. The well ordered conformation of the helix-helix connector indicates a defined interaction with lipidic surfaces, suggesting that, when bound to larger diameter synaptic vesicles, it can act as a switch between this structure and a previously proposed uninterrupted helix.

Effect of dielectric properties of solvents on the quality factor for a beyond 900 MHz cryogenic probe model

A previous report by Kelly et al. [J. Am. Chem. Soc. 124 (2002) 12013] indicated that the ionic conductivity of aqueous solution produces a significant contribution to the sensitivity loss in high-resolution NMR equipped with a cryogenically cooled probe. The loss in a sample solution contains two contributions: one from the ionic conductivity and the other from the dielectric loss; the latter is especially important at high frequencies such as above 900 MHz. Here, we investigated the effect of the dielectric conductivity on the quality factor of a 930 MHz cryogenic probe model; in particular, it deals with the ionic aqueous solutions and organic solvents commonly used for NMR in biological research and the chemistry of natural compounds. The sample quality factor, Q s, at first increases with the real part of the relative dielectric permittivity ε′ and then saturates. In the case of polar organic solvents, the transverse electric field on the sample decreases with ε′, resulting in an increase of Q s. In the case of non-polar organic solvents, the dielectric conductivity is so small that the gradient of the increase is steep, resulting in much larger Q s though the ε′ is small. The effect of the transverse electric field is negligible if the ε′ becomes large, thus the loss for ionic aqueous solution is mainly governed by a loop current induced in the sample solution. As the induced electromotive force is independent of the ε′, the Q s is saturated at high values of ε′. Based on the Q s obtained with the cryogenic probe model, the sensitivity for the cryogenic probe is expected to be as follows: the loss in sensitivity by loading water is more than 66%, i.e., the effect of the dielectric conductivity of water is remarkable at high frequencies; polar organic solvent suffers much larger losses, which is due to the enhancement of the effective sample resistance by the effect of ε′; a non-polar organic solvent is nearly free of the sensitivity loss as the dielectric conductivity is negligible; the reverse micelle behaves similarly.

Developments in Hyphenated Spectroscopic Methods in Natural Product Profiling

Overviews of the principal techniques that have found application in the profiling of natural products are given, including advances in capillary and column trapping LC-NMR-MS. Single hyphenated spectroscopic techniques, like LC-NMR and LC-MS, currently offer robust and efficient approaches for the rapid dereplication of natural product extracts. Multiple hyphenation techniques, such as (HP)LC-UV-NMR-MS-FTIR, now give an effective and comprehensive method for the deconvolution of complex mixtures. Ongoing improvements include miniaturization and cryogenic NMR probes and the hyphenation to capillary scale LC separations to analyse smaller quantities of samples. Extensions in hyphenation techniques required for natural product and other drug discovery programs include the need for LC-13C NMR and the combination of bioassays with already well-established hyphenated separationspectroscopic techniques, coupled with automated database searching capabilities (data libraries for LC, UV, NMR, MS and other search criteria).

Laser-Excited Time-Resolved Shpol'skii Spectroscopy for the Direct Analysis of Dibenzopyrene Isomers in Liquid Chromatography Fractions

We present a unique method for the unambiguous determination of dibenzo[a,l]pyrene, dibenzo[a,e]pyrene, dibenzo[a,h]pyrene, dibenzo[a,i]pyrene, and dibenzo[e,l]pyrene in high-performance liquid chromatography (HPLC) fractions. Chemical analysis is performed via laser-excited time-resolved Shpol'skii spectroscopy with the aid of a cryogenic fiber-optic probe, pulsed tunable dye laser, spectrograph, and intensified charge-coupled device. Unambiguous identification is accomplished via wavelength time matrix formats, which give simultaneous access to spectral and lifetime information. Prior to spectroscopic analysis, HPLC fractions are pre-treated with liquid-liquid extraction or solid-liquid extraction at the tip of the fiber-optic probe. Solid-liquid extraction gives the best limits of detection, which vary from 40 pg mL(-1) (dibenzo[a,l]pyrene) to 0.2 ng mL(-1)(dibenzo[e,l]pyrene).

Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe

In this study, the hyphenation of LC-SPE-NMR-MS at 500 MHz was applied to the structural elucidation of a low concentrated paracetamol metabolite present in human urine. Single or multiple peak trapping of the mass detected metabolite on SPE cartridges was employed to increase the sensitivity and quality NMR measurement over the conventional LC-NMR method. After the elution of the metabolite from the SPE cartridge to the NMR flow probe using deuterated acetonitrile for initial NMR investigation, the fraction was revovered by flushing the sample out of the NMR probe head with nitrogen gas. On the recovered fraction, high resolution FT-ICR-MS measurements were conducted, giving exact mass information about the unknown metabolite. In addition, a cryogenic NMR micro probe head was used to enhance the sensitivity of the NMR measurement by a factor of 5 in order to run 2D experiments for structural elucidation of the unknown metabolite. The combination of both MS and NMR results, led unequivocally to the elucidation of the structure as the ether glucuronide of 3-methoxyparacetamol.

Characterization of a paracetamol metabolite using on-line LC-SPE-NMR-MS and a cryogenic NMR probe

In this study, the hyphenation of LC-SPE-NMR-MS at 500 MHz was applied to the structural elucidation of a low concentrated paracetamol metabolite present in human urine. Single or multiple peak trapping of the mass detected metabolite on SPE cartridges was employed to increase the sensitivity and quality NMR measurement over the conventional LC-NMR method. After the elution of the metabolite from the SPE cartridge to the NMR flow probe using deuterated acetonitrile for initial NMR investigation, the fraction was revovered by flushing the sample out of the NMR probe head with nitrogen gas. On the recovered fraction, high resolution FT-ICR-MS measurements were conducted, giving exact mass information about the unknown metabolite. In addition, a cryogenic NMR micro probe head was used to enhance the sensitivity of the NMR measurement by a factor of 5 in order to run 2D experiments for structural elucidation of the unknown metabolite. The combination of both MS and NMR results, led unequivocally to the elucidation of the structure as the ether glucuronide of 3-methoxy-paracetamol.

Performance of Large-Size Superconducting Coil in 0.21T MRI System

A high-temperature superconductor (HTS) was used on magnetic resonance imaging (MRI) receiver coils to improve image quality because of its intrinsic low electrical resistivity. Typical HTS coils are surface coils made of HTS thin-film wafers. Their applications are severely limited by the field of view (FOV) of the surface coil configuration, and the improvement in image quality by HTS coil is also reduced as the ratio of sample noise to coil noise increases. Therefore, previous HTS coils are usually used to image small in vitro samples, small animals, or peripheral human anatomies. We used large-size HTS coils (2.5-, 3.5-, and 5.5-in mean diameter) to enhance the FOV and we evaluated their performance through phantom and human MR images. Comparisons were made among HTS surface coils, copper surface coils, and cool copper surface coils in terms of the signal-to-noise ratio (SNR) and sensitivity profile of the images. A theoretical model prediction was also used to compare against the experimental result. We then selected several human body parts, including the wrist, feet, and head, to illustrate the advantage of HTS coil over copper coil when used in human imaging. The results show an SNR gain of 200% for 5.5-in HTS coil versus same size copper coils, while for 2.5- and 3.5-in coils it is 250%. We also address the various factors that affect the performance of large size HTS coils, including the coil-to-sample spacing due to cryogenic probe and the coil-loading effect.

A phase cycle scheme that significantly suppresses offset-dependent artifacts in the R2-CPMG 15N relaxation experiment

R 2-CPMG 15N relaxation experiments form the basis of NMR dynamics measurements, both for analysis of nano–pico second dynamics and milli–micro second dynamics (kinetics). It has been known for some time that in the practical limit of finite pulse widths, which becomes acute when using cryogenic probes, systematic errors in the apparent R 2 relaxation behavior occur for spins far off-resonance from the RF carrier. Inaccurate measurement of R 2 rates propagates into quantitative models such as model-free relaxation analysis, rotational diffusion tensor analysis, and relaxation dispersion. The root of the problem stems from evolution of the magnetization vectors out of the XY-plane, both during the pulses as well as between the pulses. These deviations vary as a function of pulse length, number of applied CPMG pulses, and CPMG inter-pulse delay. Herein, we analyze these effects in detail with experimentation, numerical simulations, and analytical equations. Our work suggests a surprisingly simple change in the phase progression of the CPMG pulses, which leads to a remarkable improvement in performance. First, the applicability range of the CPMG experiment is increased by a factor of two in spectral width; second, the dynamical/kinetic processes that can be assessed are significantly extended towards the slower time scale; finally, the robustness of the relaxation dispersion experiments is greatly improved.

Microwave design and characterization of a cryogenic dip probe for time-domain measurements of nanodevices

We present microwave (MW) design, characterization, and modeling of a dip probe that can deliver ultrashort electrical pulses to semiconductor nanodevices at cryogenic temperatures (T). Systematic MW measurements, equivalent circuit modeling, and parameter extraction of the superconducting coaxial cable, in which there is a T gradient from 300 to 4.2 K, are successfully performed. De-embedding of the MW sample mounting stage (SMS) characteristic from measurement results of the dip probe dipped in liquid He is achieved using this cable modeling. The de-embedded 4.2 K characteristic of the MW SMS is in reasonable agreement with the result of three-dimensional field simulation. Finally, transmission of short electrical pulses from the pulse generator at 300 K to the MW SMS at 4.2 K, with minimal degradation, is successfully demonstrated using our dip probe. Our design and characterization technique can be applied to almost all cryogenic equipment for MW characterization.

Mechanism of Insulin Fibrillation: THE STRUCTURE OF INSULIN UNDER AMYLOIDOGENIC CONDITIONS RESEMBLES A PROTEIN-FOLDING INTERMEDIATE

Insulin undergoes aggregation-coupled misfolding to form a cross-beta assembly. Such fibrillation has long complicated its manufacture and use in the therapy of diabetes mellitus. Of interest as a model for disease-associated amyloids, insulin fibrillation is proposed to occur via partial unfolding of a monomeric intermediate. Here, we describe the solution structure of human insulin under amyloidogenic conditions (pH 2.4 and 60 degrees C). Use of an enhanced sensitivity cryogenic probe at high magnetic field avoids onset of fibrillation during spectral acquisition. A novel partial fold is observed in which the N-terminal segments of the A- and B-chains detach from the core. Unfolding of the N-terminal alpha-helix of the A-chain exposes a hydrophobic surface formed by native-like packing of the remaining alpha-helices. The C-terminal segment of the B-chain, although not well ordered, remains tethered to this partial helical core. We propose that detachment of N-terminal segments makes possible aberrant protein-protein interactions in an amyloidogenic nucleus. Non-cooperative unfolding of the N-terminal A-chain alpha-helix resembles that observed in models of proinsulin folding intermediates and foreshadows the extensive alpha --> beta transition characteristic of mature fibrils.

Modifying the surface electronic properties of YBa2Cu3O7−δ with cryogenic scanning probe microscopy

We report the results of a cryogenic study of the modification of YBa2Cu3O7−δ surface electronic properties with the probe of a scanning tunnelling microscope (STM). A negative voltage applied to the sample during STM tunnelling is found to modify locally the conductance of the native degraded surface layer. When the degraded layer is removed by etching, the effect disappears. An additional surface effect is identified using scanning Kelvin probe microscopy in combination with STM. We observe reversible surface charging for both etched and unetched samples, indicating the presence of a defect layer even on a surface never exposed to air.

Two new asterosaponins from the starfish Asterias rubens: application of a cryogenic NMR probe head

AbstractWe report two new asterosaponins from the Baltic starfish Asterias rubens along with their 1H and 13C NMR data. The compounds were isolated after on‐flow liquid chromatography–NMR–mass spectrometry screening indicated that they had not been identified before. The one‐ and two‐dimensional NMR experiments used to elucidate the structures were recorded using a 5 mm cryogenic probe head. The advantages of cryogenic probes for this kind of examination in comparison with conventional probe heads are discussed. Copyright © 2003 John Wiley & Sons, Ltd.

LC-UV-solid-phase extraction-NMR-MS combined with a cryogenic flow probe and its application to the identification of compounds present in Greek oregano.

Structure elucidation of natural products usually relies on a combination of NMR spectroscopy with mass spectrometry whereby NMR trails MS in terms of the minimum sample amount required. In the present study, the usefulness of on-line solid-phase extraction (SPE) in LC-NMR for peak storage after the LC separation prior to NMR analysis is demonstrated. The SPE unit allows the use of normal protonated solvents for the LC separation and fully deuterated solvents for flushing the trapped compounds to the NMR probe. Thus, solvent suppression is no longer necessary. Multiple trapping of the same analyte from repeated LC injections was utilized to solve the problem of low concentration and to obtain 2D heteronuclear NMR spectra. In addition, a combination of the SPE unit with a recently developed cryoflow NMR probe and an MS was evaluated. This on-line LC-UV-SPE-NMR-MS system was used for the automated analysis of a Greek oregano extract. Combining the data provided by the UV, MS, and NMR spectra, the flavonoids taxifolin, aromadendrin, eriodictyol, naringenin, and apigenin, the phenolic acid rosmarinic acid, and the monoterpene carvacrol were identified. This automated technique is very useful for natural product analysis, and the large sensitivity improvement leads to significantly reduced NMR acquisition times.

Perspectives with cryogenic RF probes in biomedical MRI

Since discovery of high-temperature superconductive (HTS) ceramics by Bednorz and Muller in 1986, there has been an accelerated development of cold technologies in industry, including the domain of NMR detection. The purpose of this paper is to fix ideas about the stage that cryogenic radio frequency (RF) probe techniques have reached in biomedical magnetic resonance imaging (MRI). Readers confronted to the literature about this emerging topic have to understand a large range of motivations with somewhat unclearly defined technical limitations and actual outlets. An overview of sensitivity issues in the general context of biomedical MRI is provided here and the contribution of RF coil techniques to recent advances is identified. The domains where cooled coil materials such as copper, low- or high-temperature superconductors, could actually increase the RF coil sensitivity are delimited by a quantitative analysis of noise mechanisms. Technical keys, cryogenic means and cold RF coil technologies are considered, and first achievements in different fields of biomedical MRI are reviewed. This survey provides a basis for discussing about the future impact of cryogenic probes for MRI investigations.

Quindolinocryptotackieine: the elucidation of a novel indoloquinoline alkaloid structure through the use of computer‐assisted structure elucidation and 2D NMR

AbstractNumerous indoloquinoline alkaloid structures have been identified from extracts of the West African plant Cryptolepis sanguinolenta. Recently, through the use of 2D NMR methods and cryogenic NMR probe technology in conjunction with computer‐assisted structure elucidation (CASE) methods, the structures of some chemical degradation products of this family of alkaloids have also been reported. We now report the characterization of a novel indoloquinoline dimeric alkaloid, quindolinocryptotackieine, through the extensive utilization of CASE methods. The NMR data presented here were collected over a decade earlier before the elucidation of the structure was possible, since manual analysis did not present a conclusive structure, whereas CASE produced a series of structures from which the structure could be verified. The original mass spectrometric (MS) data collected for the sample were problematic. Contemporary MS data were instead recollected from remaining small quantities of this alkaloid using modern instrumentation. The re‐collected data gave a usable molecular ion and several key fragment ions that were diagnostically useful. Copyright © 2003 John Wiley & Sons, Ltd.

LTS SQUID microscope with micron spatial resolution

We describe a multichannel LTS SQUID microscope with micron spatial resolution. The system achieves micron resolution by the use of small (14 /spl mu/m) detection coils and narrow gap between the coils and the object(s) being scanned. Samples are mounted inside an exchange-gas can at the lower end of a cryogenic probe. This houses all of the cryogenic portions of the microscope and is filled with helium exchange gas. A 5 mm/spl times/5 mm scanning stage is used to scan the sample, which is at cryogenic temperatures. Stepping motors on the scanning stage allow step sizes as small as 0.16 /spl mu/m. The SQUIDs are mounted on a cantilever structure with nine separate detection coils on the end of the structure. Flux noise of the SQUIDs is better than 5 /spl mu//spl Phi//sub o///spl radic/Hz. Sensitivity is better than 100 pT//spl radic/Hz with a bandwidth of dc-10 kHz. Open architecture software provides control of all critical system components, along with data acquisition and analysis. We have demonstrated spatial resolutions better than 2 /spl mu/m. We discuss the impact of the external field coils for susceptibility measurements.

Shpol'skii spectroscopy at the interface of two non-polar microenvironments: a novel approach for the analysis of polycylic aromatic compounds

This article presents a thorough investigation of quantitative parameters for the analysis of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs) and polychlorinated dibenzofurans (PDBFs) partitioning between an octadecyl silica extraction membrane and microliters of an n-alkane solvent spiked on the surface of the membrane. Shpol'skii spectrometry is directly performed on the surface of the membrane with the aid of a cryogenic fiber optic probe. The analyte concentration in the layer of Shpol'skii solvent follows a linear relationship with the analyte concentration in the water sample and the same is observed for the phosphorescence signal of the cryogenic probe. The accuracy and precision needed for quantitative analysis in aqueous samples is demonstrated. The analytical figures of merit show the feasibility to determine organic pollutants at the parts-per-trillion level with minimum solvent consumption.

Analysis of polycyclic aromatic hydrocarbons in HPLC fractions by laser-excited time-resolved Shpol'skii spectrometry with cryogenic fiber-optic probes

Laser-excited time-resolved Shpol'skii spectrometry at liquid helium temperature (4.2 K) is presented for the analysis of polycyclic aromatic hydrocarbons in high-performance liquid chromatography fractions. Fluorescence measurements are rapidly done with the aid of a fiber-optic probe, pulsed tunable dye laser, spectrograph, and intensified charge-coupled device. Analyte identification and peak-purity checking are made through wavelength–time matrix formats, which give simultaneous access to spectral and lifetime information. Sample preparation is rapid and simple. It involves liquid–liquid extraction or solid–liquid extraction of chromatographic fractions at the tip of the fiber-optic probe. The potential of both approaches is demonstrated with the semi-quantitative analysis of priority pollutants in heavily contaminated water samples.

Advancing NMR sensitivity for LC-NMR-MS using a cryoflow probe: application to the analysis of acetaminophen metabolites in urine.

Cryogenic cooling of the NMR radio frequency coils and electronics to give greatly enhanced sensitivity is arguably the most significant recent advance in NMR spectroscopy. Here we report the first cryogenic probe built in flow configuration and demonstrate the application to LC-NMR-MS studies. This probe provides superior sensitivity over conventional noncryogenic flow NMR probes, allowing the use of 100 microL of untreated urine (40% less material than previous studies that required preconcentration) and yet revealing drug metabolites hitherto undetected by LC-NMR-MS at 500 MHz. Besides the known sulfate and glucuronide metabolites, previously undetected metabolites of acetaminophen were directly observable in a 15-min on-flow experiment. Simultaneous MS data also provided knowledge on the NMR-silent functional moieties. Further, stop-flow LC-NMR-MS experiments were conducted for greater signal-to-noise ratios on minor metabolites. The cryoflow probe enables the NMR analysis of lower concentrations of metabolites than was previously possible for untreated biofluids. This strategy is generally applicable for samples containing mass-limited analytes, such as those from drug metabolism studies, biomarker and toxicity profiling, impurity analysis, and natural product analysis.