Label-free quantitative detection of protein using macroporous silicon photonic bandgap biosensors

A label-free biosensor was demonstrated using macroporous silicon (pore size >100 nm) one-dimensional photonic band gap structures that are very sensitive to refractive index changes. In this study, we employed Tir-IBD (translocated Intimin receptor-Intimin binding domain) and Intimin-ECD (extracellular domain of Intimin) as the probe and target, respectively. These two recombinant proteins comprise the extracellular domains of two key proteins responsible for the pathogenicity of enteropathogenic Escherichia coli (EPEC). The optical response of the sensor was characterized so that the capture of Intimin-ECD could be quantitatively determined. Our result shows that the concentration sensitivity limit of the sensor is currently 4 microM of Intimin-ECD. This corresponds to a detection limit of approximately 130 fmol of Intimin-ECD. We have also investigated the dependence of the sensor performance on the Tir-IBD probe molecule concentration and the effect of immobilization on the Tir-IBD/Intimin-ECD equilibrium dissociation constant. A calibration curve generated from purified Intimin-ECD solutions was used to quantify the concentration of Intimin-ECD in an E. coli BL21 bacterial cell lysate, and results were validated using gel electrophoresis. This work demonstrates for the first time that a macroporous silicon microcavity sensor can be used to selectively and quantitatively detect a specific target protein with micromolar dissociation constant (Kd) in a milieu of bacterial proteins with minimal sample preparation.     

Canine cancer screening via ultraviolet absorbance and fluorescence spectroscopy of serum proteins

A cost-effective optical cancer screening and monitoring technique was demonstrated in a pilot study of canine serum samples and was patented for commercialization. Compared to conventional blood chemistry analysis methods, more accurate estimations of the concentrations of albumin, globulins, and hemoglobin in serum were obtained by fitting the near UV absorbance and photoluminescence spectra of diluted serum as a linear combination of component reference spectra. Tracking these serum proteins over the course of treatment helped to monitor patient immune response to carcinoma and therapy. For cancer screening, 70% of dogs with clinical presentation of cancer displayed suppressed serum hemoglobin levels (below 20 mg/dL) in combination with atypical serum protein compositions, that is, albumin levels outside of a safe range (from 4 to 8 g/dL) and globulin levels above or below a more normal range (from 1.7 to 3.7 g/dL). Of the dogs that met these criteria, only 20% were given a false positive label by this cancer screening test.     

Ionizable isotopic labeling reagent for relative quantification of amine metabolites by mass spectrometry

A powerful approach to relative quantification by mass spectrometry is to employ labeling reagents that target specific functional groups in molecules of interest. A quantitative comparison of two or more samples may be readily accomplished by using a chemically identical but isotopically distinct labeling reagent for each sample. The samples may then be combined, subjected to purification steps, and mass analyzed. Comparison of the signal intensities obtained from the isotopically labeled variants of the target analyte(s) provides quantitative information on their relative concentrations in the sample. In this report, we describe the synthesis and use of heavy and light isotopic forms of methyl acetimidate for the relative quantification of amine-containing species. The principal advantages of methyl acetimidate as a labeling reagent are that the reaction product is positively charged and hydrophobicity is increased, both of which enhance electrospray ionization efficiency and increase detection sensitivity. The quantitative nature of the analysis was demonstrated in model metabolomics experiments in which heavy and light labeled Arabidopsis extracts were combined in different ratios. Finally, the labeling strategy was employed to determine differences in the amounts of amine-containing metabolites for Arabidopsis seeds germinated under two different conditions.     

Two-wave-plate compensator method for full-field retardation measurements

The two-wave-plate compensator (TWC) method is expanded for full-field retardation measurements by use of a polarization microscope. The sample image is projected onto a CCD camera connected to a computer, allowing the retardation to be measured at all pixels. The retardation accuracy of this implementation of the TWC is evaluated to be 0.06 nm. The method is applied to polarization-maintaining fibers and long-period fiber gratings. The measured retardation is in good agreement with the crossed-polarizer images of the fibers. The method achieves a spatial resolution of 0.45 microm and a retardation resolution of 0.07 nm. The full-field TWC method can thus be a useful tool for characterizing and monitoring the fabrication of optical devices.     

Enhanced stability of recombinant keratinocyte growth factor by mutagenesis

Native sequence keratinocyte growth factor (KGF) is fairly unstable, as manifested by the loss of the monomeric native protein accompanied by the accumulation of aggregated species during storage at moderate temperatures. Several different types of analogs were generated and the storage stability of the protein assessed. In the first type of analog one or more of the five cysteinyl residues in KGF were replaced; in the second class the N-terminal residues that included the first disulfide bond were deleted. Both of these types of analogs involved removal of the disulfide bond between cysteines 1 and 15. The third group involved mutating one of the basic amino acids located in a cluster of positive charges (involved in heparin binding) around Arg144 to a neutral or acidic amino acyl residue. Among the cysteine replacement analogs, the double mutation of Cys1 and 15 to Ser resulted in significantly increased stability without compromising the mitogenic activity, while Cys to Ser mutations at other positions were either destabilizing or had no effect. Deletion of the 15, 23 or 27 N-terminal amino acyl residues also increased the stability of the protein. The activity of the analogs was not affected by the deletion of 15 or 23 amino acids, but it was significantly decreased upon removal of the 27 N-terminal amino acyl residues. Much greater stability was achieved by mutation of the basic amino acids, especially Arg144, to Glu or Gln, but this increase in stability was accompanied by large decrease in activity. The analog with the 23 N-terminal amino acyl residues deleted represents one of the best compromises between increased stability and retention of activity.     

Large thermoelectric figure-of-merits from SiGe nanowires by simultaneously measuring electrical and thermal transport properties

The strongly correlated thermoelectric properties have been a major hurdle for high-performance thermoelectric energy conversion. One possible approach to avoid such correlation is to suppress phonon transport by scattering at the surface of confined nanowire structures. However, phonon characteristic lengths are broad in crystalline solids, which makes nanowires insufficient to fully suppress heat transport. Here, we employed Si-Ge alloy as well as nanowire structures to maximize the depletion of heat-carrying phonons. This results in a thermal conductivity as low as ～1.2 W/m-K at 450 K, showing a large thermoelectric figure-of-merit (ZT) of ～0.46 compared with those of SiGe bulks and even ZT over 2 at 800 K theoretically. All thermoelectric properties were "simultaneously" measured from the same nanowires to facilitate accurate ZT measurements. The surface-boundary scattering is prominent when the nanowire diameter is over ～100 nm, whereas alloying plays a more important role in suppressing phonon transport for smaller ones.     

Control of lateral dimension in metal-catalyzed germanium nanowire growth: usage of carbon sheath

We report on the catalytic growth of thin carbon sheathed single crystal germanium nanowires (GeNWs), which can solve the obstacles that have disturbed a wide range of applications of GeNWs. Single crystal Ge NW core and amorphous carbon sheath are simultaneously grown via vapor-liquid-solid (VLS) process. The carbon sheath completely blocks unintentional vapor deposition on NW surface, thus ensuring highly uniform diameter, dopant distribution, and electrical conductivity along the entire NW length. Furthermore, the sheath not only inhibits metal diffusion but also improves the chemical stability of GeNWs at even high temperatures.     

Metastable Ge1-xCx alloy nanowires

Carbon-containing alloy materials such as Ge(1-x)C(x) are attractive candidates for replacing silicon (Si) in the semiconductor industry. The addition of carbon to diamond lattice not only allows control over the lattice dimensions, but also enhances the electrical properties by enabling variations in strain and compositions. However, extremely low carbon solubility in bulk germanium (Ge) and thermodynamically unfavorable Ge-C bond have hampered the production of crystalline Ge(1-x)C(x) alloy materials in an equilibrium growth system. Here we successfully synthesized high-quality Ge(1-x)C(x) alloy nanowires (NWs) by a nonequilibrium vapor-liquid-solid (VLS) method. The carbon incorporation was controlled by NW growth conditions and the position of carbon atoms in the Ge matrix (at substitutional or interstitial sites) was determined by the carbon concentration. Furthermore, the shrinking of lattice spacing caused by substitutional carbon offered the promising possibility of band gap engineering for photovoltaic and optoelectronic applications.     

A novel nano-configuration for thermoelectrics: helicity induced thermal conductivity reduction in nanowires

In this article, we propose a novel helical nano-configuration towards the designing of high ZT thermoelectric materials. Non-equilibrium molecular dynamics (NEMD) simulations for 'model' bi-component nanowires indicate that a significant reduction in thermal conductivity, similar to that of flat superlattice nanostructures, can be achieved using a helical geometric configuration. The reduction is attributed to a plethora of transmissive and reflective phonon scattering events resulting from the steady alteration of phonon propagating direction that emerges from the continuous rotation of the helical interface. We also show that increasing the relative mass ratio of the two components lowers the phonon energy transmission at the interface due to differences in vibrational frequency spectra, thereby relatively 'easing' the phonon energy propagation along the helical pathway. While the proposed mechanisms result in a reduced lattice thermal conductivity, the continuous nature of the bi-component nanowire would not be expected to significantly reduce its electrical counterpart, as often occurs in superlattice/alloy nanostructures. Hence, we postulate that the helical configuration of atomic arrangement provides an attractive and general framework for improved thermoelectric material assemblies independent of the specific chemical composition.     

Incidence and severity of Arcanobacterium pyogenes injection site abscesses with needle or needle-free injection

Nursery-age pigs (n=198) were used to evaluate the difference in abscess formation at needle-free jet and conventional needle-and-syringe injection sites. Needle-free jet injection was used to administer injections in the neck and ham on one side of the animal whereas needle-and-syringe was used for neck and ham injections on the opposite side. Immediately prior to injection, the injection site surfaces were contaminated with an inoculum of Arcanobacterium pyogenes. Each pig was humanely euthanized 27 or 28days after injections. Histopathological results showed that needle-free jet injection was associated with more abscesses than needle-and-syringe injection at both neck (P=0.0625) and ham (P=0.0313) injection sites. Out of 792 injection sites, only 13 abscesses were observed, with 12 of those present at needle-free jet injection sites. Needle-free jet injection may increase the occurrence of injection site abscesses that necessitate carcass trimming at pork processing plants.