Microfabricated bioreactor chips for immobilised enzyme assays

A microfabricated device is reported that has been designed to permit the in situ packing of a section of channel with enzyme immobilised onto controlled pore glass (CPG). It is fabricated from glass and polydimethylsiloxane and to prevent dead volumes, has dedicated channels for packing the reactor. The device has the advantage of being simple in design, the flow through enzyme reactor channel being simply a widened section of the analyte channel. The system is suitable for both hydrodynamic and electro-osmotic pumping, and is designed such that when the packing is exhausted it can be repacked. Controlled pore glass provides a reproducible none swelling, high porosity medium onto which the enzyme could be immobilised. The large particle size meant that it was vital to optimise the immobilisation procedure in order to achieve acceptable enzyme activity. The microfabricated device was developed with two enzymes of different molecular masses; alkaline phosphatase and xanthine oxidase. The pore size of the CPG was found to be very important for xanthine oxidase, where the 697 Å pore size (120-200 mesh) CPG was found to give the highest activity (18-20% activity retained after immobilisation). The microfabricated device was used for the assay of p-nitrophenyl phosphate and hypoxanthine with spectrophotometric detection at 405 and 470 nm, respectively. The limits of detection were 5 and 8 μM, respectively.     

对一个大单轴磁各向异性镍(Ⅱ)配合物中的磁跃迁的直接观察

最近Ruamps和同事发现三角双锥构型的Ni（Ⅱ）配合物[Ni（Me₆tren）Cl]ClO₄（1，Me₆tren=tris（2-（dimethylamino）ethyl）amine）具有大的单轴磁各向异性（J.Am.Chem.Soc.，2013，135：3017-3026）。他们利用HF-EPR研究获得横向零场分裂（ZFS）参数E=1.56（5）cm^-1但未能确定轴向零场分裂参数D。在本工作中，我们利用0~17.5 T和5 K的变磁场远红外光谱（FIRMS）来检测自旋基态S=1中的M_S=±1和M_S=0态之间的磁跃迁。在FIRMS中直接观察到Zeeman分裂态之间的跃迁，得出轴向ZFS参数D=-110.7（3）cm^-1。我们对1的晶体结构进行了Hirshfeld表面分析，揭示了1分子中的阳离子与阴离子之间以及分子之间的相互作用。     

Spectral baseline ripple in hot-electron bolometer mixers due to local oscillator phase and amplitude instabilities

High resolution millimetre and submillimetre wave astronomical spectrometers using hot-electron bolometer mixers as detectors often show marked standing wave patterns in the spectral baseline. LO phase noise contributes through two mechanisms: the phase noise side-bands may be converted to amplitude noise in the source because of the power-frequency characteristic of the source, or they can be converted to in-band amplitude noise through the action of the quasi-optical discriminator formed by the mixer, beam-splitter and telescope structure. The baseline ripple components due to each of these mechanisms have different characteristic periods, and under some circumstances can dominate the spectrometer baseline. The ripple levels estimated using the theory agree well with those observed in practice. It is shown that with careful design systematic effects due to this cause can be reduced to a negligible level.     

VSWR reduction for large millimeter-wave cassegrain radiotelescopes

Multiple reflections in large radiotelescopes used for astronomical spectroscopy cause characteristic modulations of the observed spectrum (baseline ripple). For a given mechanism, the magnitude of the effect depends primarily on the reflection coefficient, which for the most important paths is proportional to . Although ripple is thus generally negligible at mm-wavelengths, there are some instances where it may still be significant, and a recent experiment at the15 m James Clerk Maxwell Telescope was severely affected. We describe a technique which has proved effective in reducing the ripple to an acceptable level.The purpose of this research note is to draw this technique to the attention of the astronomical community. We calculate the reflection coefficient for a typical instrument and show that it can be greatly reduced over a very broad bandwidth by means of a tapered absorber in the centre of the secondary mirror. The graphical analysis of the tapered absorber also demonstrates that the scattering cones and circular absorbers widely used in radioastronomy donot in fact reduce the reflection coefficient significantly.     

Tuning Tetrazole Photochemistry for Protein Ligation and Molecular Imaging

Photochemistry provides a wide range of alternative reagents that hold potential for use in bimolecular functionalisation of proteins. Here, we report the synthesis and characterisation of metal ion binding chelates derivatised with disubstituted tetrazoles for the photoradiochemical labelling of monoclonal antibodies (mAbs). The photophysical properties of tetrazoles featuring extended aromatic systems and auxochromic substituents to tune excitation toward longer wavelengths (365 and 395 nm) were studied. Two photoactivatable chelates based on desferrioxamine B (DFO) and the aza-macrocycle NODAGA were functionalised with a tetrazole and developed for protein labelling with ⁸⁹Zr, ⁶⁴Cu and ⁶⁸Ga radionuclides. DFO-tetrazole ( 1 ) was assessed by direct conjugation to formulated trastuzumab and subsequent radiolabelling with ⁸⁹Zr. Radiochemical studies and cellular-based binding assays demonstrated that the radiotracer remained stable in vitro retained high immunoreactivity. Positron emission tomography (PET) imaging and biodistribution studies were used to measure the tumour specific uptake and pharmacokinetic profile in mice bearing SK-OV-3 xenografts. Experiments demonstrate that tetrazole-based photochemistry is a viable approach for the light-induced synthesis of PET radiotracers.     

Water suppression without signal loss in HR-MAS 1H NMR of cells and tissues

In cell and tissue samples, water is normally three orders of magnitude more abundant than other metabolites. Thus, water suppression is required in the acquisition of NMR spectra to overcome the dynamic range problem and to recover metabolites that overlap with the broad baseline of the strong water resonance. However, the heterogeneous cellular environment often complicates water suppression and the strong coupling of water to membrane lipids interferes with the NMR detection of membrane associated lipid components. The widely used water suppression techniques including presaturation and double pulsed field gradient selective echo result in more than a 70% reduction in membrane associated lipid components in proton spectra of cells and tissues compared to proton spectra acquired in the absence of water suppression. A water suppression technique based on the combination of selective excitation pulses and pulsed field gradients is proposed to use in the acquisition of high resolution MAS NMR spectra of tissue specimens and cell samples. This pulse sequence methodology enables efficient water suppression for intact cells and tissue samples and eliminates signal loss from cellular metabolites.     

Llama-derived single-domain antibodies for the detection of botulinum A neurotoxin

Single-domain antibodies (sdAb) specific for botulinum neurotoxin serotype A (BoNT A) were selected from an immune llama phage display library derived from a llama that was immunized with BoNT A toxoid. The constructed phage library was panned using two methods: panning on plates coated with BoNT A toxoid (BoNT A Td) and BoNT A complex toxoid (BoNT Ac Td) and panning on microspheres coupled to BoNT A Td and BoNT A toxin (BoNT A Tx). Both panning methods selected for binders that had identical sequences, suggesting that panning on toxoided material may be as effective as panning on bead-immobilized toxin for isolating specific binders. All of the isolated binders tested were observed to recognize bead-immobilized BoNT A Tx in direct binding assays, and showed very little cross-reactivity towards other BoNT serotypes and unrelated protein. Sandwich assays that incorporated selected sdAb as capture and tracer elements demonstrated that all of the sdAb were able to recognize soluble (“live”) BoNT A Tx and BoNT Ac Tx with virtually no cross-reactivity with other BoNT serotypes. The isolated sdAb did not exhibit the high degree of thermal stability often associated with these reagents; after the first heating cycle most of the binding activity was lost, but the portion of the protein that did refold and recover antigen-binding activity showed only minimal loss on subsequent heating and cooling cycles. The binding kinetics of selected binders, assessed by both an equilibrium fluid array assay as well as surface plasmon resonance (SPR) using toxoided material, gave dissociation constants (K _D ) in the range 2.2 × 10⁻¹¹ to 1.6 × 10⁻¹⁰ M. These high-affinity binders may prove beneficial to the development of recombinant reagents for the rapid detection of BoNT A, particularly in field screening and monitoring applications.