Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (K_D) of a protein–antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled (“conformational”) antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA–aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.     

Structure-activity relationship studies on the immune stimulatory effects of base-modified CpG toll-like receptor 9 agonists

Synthetic oligodeoxynucleotides containing unmethylated deoxycytidylyl-deoxyguanosine dinucleotide (CpG) motifs are able to stimulate potent immune responses through a signaling pathway involving Toll-like receptor 9 (TLR9). We have investigated the structure-activity relationship (SAR) of base-modified CpG oligonucleotides with TLR9 by measuring TLR9 activation by 20-mer oligonucleotides having just a single human recognition motif (5'-GTCGTT-3') in functional cell-based TLR9 assays. Substitution of guanine by hypoxanthine and 6-thioguanine resulted in activity similar to the unmodified parent molecule, whereas purine, 2-aminopurine, 2,6-diaminopurine, and 8-oxo-7,8-dihydroguanine substitution resulted in approximately 40-60 % reduction in activity, and 7-deazaguanine substitution led to the strongest (80 %) reduction in TLR9 stimulation. Furthermore, none of the investigated modifications at C5 and N4 of cytosine were well tolerated with respect to human TLR9 stimulation. Our results are compatible with a SAR model in which guanine is recognized by the Hoogsteen site, and C5 is most critical for recognition of cytosine. In addition, we found significant species-specific differences between human and murine TLR9 recognition, which demonstrates the importance of choosing appropriate assay systems for SAR studies.     

Effect of gadolinium ions on the structure and magnetic properties of zinc-borate glasses and glass ceramics

X-ray diffraction (XRD), electron paramagnetic resonance (EPR), and magnetic susceptibility measurements have been employed to investigate the samples from the (Gd₂O₃) _x ·(B₂O₃)_(60−x)·(ZnO)₄₀ (0 ≤ x ≤ 20 mol%) system. The XRD pattern for the prepared samples shows that the vitreous phase is present only for x ≤ 15 mol%. For the samples containing 20 mol% Gd₂O₃ the presence of a unique crystalline phase, GdBO₃, embedded in a vitreous matrix was evidenced. In this case the XRD patterns show the presence of nanometer sized crystals (64 nm) in a glassy matrix. The EPR spectra of the studied samples exhibit three important features with effective g-values of ≈6, 2.8, 2.0 and a weaker feature at g ≈ 4.8. For low Gd₂O₃ contents (x < 3 mol%), the EPR spectra have the typical ‘‘U’’-type shape. For higher contents of Gd₂O₃ (x ≥ 3 mol%), the spectral features are broadened and finally are dominated by a single broad absorption line located at g ≈ 2.0. This broad EPR line is associated to the Gd³⁺ ions present predominantly as clustered species. Magnetic susceptibility data show that for x > 1 mol% the Gd³⁺ ions are present not only as isolated species but also as species coupled through antiferromagnetic exchange interactions.     

CuInS2-Poly(3-(ethyl-4-butanoate)thiophene) nanocomposite solar cells: Preparation by an in situ formation route, performance and stability issues

In this contribution we present an in situ method for the preparation of CuInS₂-poly(3-(ethyl-4-butanoate)thiophene) (P3EBT) nanocomposite layers and their application in nanocomposite solar cells. A precursor solution containing copper and indium salts, thiourea and the conjugated polymer was prepared in pyridine, which was coated onto glass/ITO substrates followed by a heating step at 180 °C. The heating step induced the formation of the CuInS₂ nanoparticles homogeneously dispersed in the conjugated polymer matrix. The formation of the nanocomposite was investigated in situ by X-ray scattering techniques and TEM methods showing that nano-scaled CuInS₂ was formed. By addition of small amounts of zinc salt to the precursor solution, zinc containing CuInS₂ (ZCIS) was formed. ZCIS-P3EBT active layers exhibited higher V_OC than CuInS₂-P3EBT layers and showed efficiencies of about 0.4%. Additionally the stability of the solar cells was tested over a time scale of 172 h.     

Fabrication and characterization of magnetic cotton yarns for textile applications

Cotton yarns covered with hard (barium hexaferrite – BaFe), and soft (Black Toner 6745 CP-313) magnetic particles are investigated for their application in the field of functional textile. Our results reveal a rather consistent dependence of the magnetic yarn properties on the fabrication parameters (nature of the magnetic powder, composition of the coating solution, etc.). The magnetic properties (residual magnetism and coercive field intensity) of the yarns are increasing with the magnetic powder content in the coating solution. However, this is at some point leading to the degradation of some yarn properties, e.g. elasticity. Bending tests revealed that several factors improve the bending behavior of the yarns and reduce cracks. For example, a higher number of cracks were exhibited by the hard magnetic yarns compared to the soft ones. The presence of polyurethane in the coating solution leads to no cracks on the surface of hard magnetic yarns or a small number in case of soft magnetic yarns. The tensile properties of the magnetic yarns are increasing with increasing magnetic powder content. Over a certain mass percentage (45% for BaFe and 30% for CP-313), the magnetic yarns tensile strength and elongation become lower than those of the support yarn.     

Oscillatory behavior of the magnetic properties of Nd–Fe–B films with Mo and Mo–Cu additions

A series of Ta/NdFeB/Ta thin films with Mo and Mo–Cu additions embedded by alloying and by stratification have been prepared by r.f. sputtering. The influence of additions, their embedding mode, and annealing temperature on the structural and magnetic behavior of Ta/NdFeB/Ta thin films is presented. The use of additions of Mo and Mo–Cu leads to refined grain structure and improvement in the hard magnetic characteristics of Ta/NdFeB/Ta thin films. The Ta/[NdFeBMo(540 nm)/Ta films and Ta/[NdFeB(180 nm)/MoCu(dnm)] × n/Ta multilayer films present enhanced coercivities and M_r/M_s ratios in comparison with the Ta/NdFeB(540 nm)/Ta films. The stratification of Ta/NdFeB/Ta thin films with Mo–Cu interlayers leads to an oscillatory behavior of hard magnetic characteristics of the Ta/[NdFeB(180 nm)/MoCu(dnm)] × n/Ta multilayer films, when the thickness, d, of Mo–Cu interlayers varies by increments of 1 nm. When the thickness of Mo–Cu interlayers varies by increments of 2 nm the oscillatory behavior of the magnetic characteristics is not revealed. For a thickness of the Mo–Cu interlayer of 3 nm in the Ta/[NdFeB(180 nm)/MoCu(3 nm)] × 3/Ta thin films annealed at 650 °C, the c-axis of part of the hard magnetic Nd₂Fe₁₄B grains is oriented out-of-plane.     

Structural properties of silver doped hydroxyapatite and their biocompatibility

The aim of this study was to obtain a novel hydroxyapatite-based material with high biocompatibility. The structural properties of the samples were well characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray Photoelectron Spectroscopy (XPS). The X-ray diffraction studies revealed the characteristic peaks of hydroxyapatite in each sample. Other phases or impurities were not observed. The scanning electron microscopy observations suggest that the doping components have no influence on the surface morphology of the samples, which reveals a homogeneous aspect of the synthesized particles for all samples. The presence of calcium (Ca), phosphor (P), oxygen (O) and silver (Ag) in the Ag:HAp is confirmed by energy dispersive X-ray (EDAX) and X-ray Photoelectron Spectroscopy analyses. Nanocrystalline silver doped HAp stimulated viability and potentiated the activation of murine macrophages.     

Investigation of Electrochemical Properties of Carbon Nanofibers Prepared by CCVD Method

Carbon nanofibers, with diameters between 80 and 290 nm and specific area of 242 m²g^?1, have been prepared by the catalytic chemical vapor deposition method. After preparation, the powder was mixed with silicon oil in order to create a paste electrode. The electrochemical behavior of this type of electrode was investigated by cyclic voltammetry, using a solution of 10^?3 M ferrocenecarboxylic acid as mediator. The redox process is quasi-reversible, and it involves the transfer of electrons between Fe(II) and Fe(III).

The same mediator was used for the construction of a second-generation glucose biosensor. The mediator was co-immobilized with the enzyme in the carbon nanofibers paste. The sensor linearly responded to glucose, in the concentration range of 1.7 to 7 mM. A time of around 30 seconds was required to reach 95% of the maximum steady-state current.

Also, the oxidation of calf thymus DNA at the carbon nanofiber paste electrode was investigated by differential pulse voltammetry (DPV). A clear signal, due to guanine oxidation, was obtained in the case of single-stranded DNA.