Quantitative biosensor detection by chemically exchanging hyperpolarized 129Xe.

Chemical sensors informing about their local environment are of widespread use for chemical analysis. A thorough understanding of the sensor signaling is fundamental to data analysis and interpretation, and a requirement for technological applications. Here, sensors explored for the recognition and display of biomolecular and cellular markers by magnetic resonance and composed of host molecules for xenon atoms are considered. These host-guest systems are analytically powerful and also function as contrast agents in imaging applications. Using nuclear spin hyperpolarization of 129Xe and chemical exchange saturation transfer the detection sensitivity is orders of magnitude enhanced in comparison to conventional 1H NMR. The sensor signaling reflects this rather complex genesis, furthering the mere qualitative interpretation of biosensing data; to harvest the potential of the approach, however, a detailed numerical account is desired. To this end, we introduce a comprehensive expression that maps the sensor detection quantitatively by integration of the hyperpolarization generation and relaxation with the host-xenon exchange dynamics. As demonstrated for the host molecule and well-established biosensor cryptophane-A, this model reveals a distinguished maximum in sensor signaling and exerts control over experimentation by dedicated adjustments of both the amount of xenon and the duration of the saturation transfer applied in a measurement, for example to capitalize on investigations at the detection limit. Furthermore, usage of the model for data analysis makes the quantification of the sensor concentration in the nanomolar range possible. The approach is readily applicable in investigations using cryptophane-A and is straightaway adaptable to other sensor designs for extension of the field of xenon based biosensing.

Characteristics of waves guided by a grounded "left-handed" material slab of finite extent.

The properties of waves guided by a plane-parallel finite slab of material having an ideal, homogeneous, and causal permittivity epsilon (f) , and permeability mu (f) , are investigated analytically and numerically through simulations done via a finite difference time domain (FDTD) code. Lorentzian functional forms are chosen for epsilon (f) and mu (f) . Wave guidance is examined for frequency ranges where the material in the slab is in the left-handed material (LHM) regime, i.e., the real parts of epsilon (f) and mu (f) are negative. It is shown that for reasonably thin slabs, and unlike ordinary materials, there is a unique power recirculation or feedback mechanism wherein the fields in the vicinity of the slab exchange power across the free-space/LHM slab interface. Within the LHM slab, the power travels backwards towards the source. This results in significant but bounded energy accumulation near the edge of the slab closest to the source. The energy exchange across the slab interface is necessary in order to sustain the resulting backward wave in the slab. Slabs thicker than a wavelength are also analyzed, leading to a reversal of the power loop description. The agreement between analytical and numerical results is excellent. They confirm the guided wave physics of a LHM slab.

Signal selection in high-resolution NMR by pulsed field gradients. I. Geometrical analysis.

A geometrical description for the selection of coherence transfer pathways in high resolution NMR by the application of pulsed field gradients along three orthogonal directions in space is presented. The response of the spin system is one point of the three-dimensional Fourier transform of the sample volume affected by a sequence of field gradients. The property that a pathway is retained (or suppressed) when a sequence of field gradients is applied is expressed by the property of vectors, representing the pathway and the sequence, respectively, to be orthogonal (or not orthogonal). Ignoring imperfections of RF pulses, and with the exception of sensitivity enhanced experiments and experiments where the relevant coherence order is zero while field gradients are applied, it is shown that at most only half of the relevant pathways, as compared to a phase cycled experiment, are retained when field gradients are used for signal selection.

Signal selection in high-resolution NMR by pulsed field gradients.

We describe a new and powerful computer program called TRIPLE GRADIENT which calculates optimized pulsed field gradient sequences for specific coherence pathway selection or rejection. Sequences can be computed for gradient coils acting along one, two, or three perpendicular axes. The program is based on the computational minimization of a penalty function formed from the summed amplitudes of the unwanted signals. The underlying mathematical analysis makes use of a vectorial representation of the way in which a gradient sequence suppresses different signals. It is argued that experiments using well-calculated gradient sequences are quicker and generally perform better than those using extensive phase cycling, especially when suppressing extremely strong solvent signals, and it is shown that in many cases gradient experiments of optimal signal-to-noise ratio can be performed. These claims are illustrated by spectra obtained from an HQQC experiment.

The structures of native phosphorylated chicken cystatin and of a recombinant unphosphorylated variant in solution.

The solution structures of the phosphorylated form of native chicken cystatin and the recombinant variant AEF-S1M-M29I-M89L were determined by 2D, 3D and 4D-NMR. The structures turn out to be very similar, despite the substitutions and the phosphorylation of the wild-type. Their dominant feature is a five-stranded beta-sheet, which is wrapped around a five-turn alpha-helix, as shown by X-ray crystallographic studies of wild-type chicken cystatin. However, the NMR analysis shows that the second helix observed in the crystal is not present in solution. The phosphorylation occurs at S80, which is located in a flexible region. For this reason, very few effects on the structure are observed. Comparison of structures of the unphosphorylated variant and the wild-type shows small effects on H84 which is located in the supposed recognition site of the serine kinase. This recognition site appears to be well structured as a large loop-containing bulge of the beta-sheet. The N termini of both mutants, which contribute to a large extent to the binding to the proteinase, are very flexible. A loop structure involving the residues L7 to A10 as found in related inhibitors, such as in the kininogen domains 2 and 3, is not sufficiently populated to be observed.

Purification and characterization of a chicken egg white cystatin variant expressed in an Escherichia coli pIN-III-ompA system.

A synthetic gene coding for a chicken egg white cystatin variant was cloned and expressed using the pIN-III-ompA Escherichia coli expression system. After osmotic shock of the E. coli cells, the cysteine proteinase inhibitor was isolated from periplasm and purified by S-carboxymethylpapain affinity chromatography. The resulting inhibitory material was characterized by SDS/PAGE, reversed-phase HPLC, peptide mapping and amino acid sequencing. The recombinant variant chicken AEF-[S1----M, M29----I, M89----L]cystatin shows strong inhibitory activity and displays Ki values in the complex with papain, actinidin and cathepsin B similar to those found for natural chicken cystatin. The purified variant showed a native-chicken-cystatin-like conformational state, as determined by NMR spectroscopy, if the NMR data of 15N-labelled recombinant inhibitor were compared with those of the natural inhibitor.