RESUMO
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 (KD) 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.
Assuntos
Anticorpos/análise , Aptâmeros de Nucleotídeos/análise , Técnicas Biossensoriais/métodos , Epitopos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Anticorpos/química , Anticorpos/imunologia , Afinidade de Anticorpos , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/imunologia , Epitopos/química , Epitopos/imunologia , Humanos , Ressonância de Plasmônio de Superfície/métodosRESUMO
We present a new principle for tuning the diffraction efficiency of an optical grating and its implementation in a micro-optical device. The overlap of two phase gratings is used to vary the effective phase shift and hence the diffraction efficiency. We study the working principle using Fourier Optics to simulate the diffraction pattern in the far field and design and realize a device based on integrated piezo actuation. We find good agreement between simulation and experiment and observe a suppression of the first diffraction order intensity by more than 97% and response times of less than 3 ms.
RESUMO
Characterization of thermoelectric transport properties for temperature sensing, cooling, and energy harvesting applications is necessary for a reliable device performance in progressively minimized computer chips. In this contribution, we present a fully automated thermovoltage and sheet resistance measurement setup, which is calibrated and tested for the production of silicon- and silicon-germanium-doped as well as silicide complementary metal-oxide-semiconductor-compatible thin films. A LabVIEW-programmed software application automatically controls the measurement and recording of thermovoltages at individually defined temperature set points. The setup maps average temperature and temperature differences simultaneously in the regime from 40 to 70 °C. The Seebeck coefficient calculated by means of the inversion method was used to eliminate the offset voltage influence. Finally, we present and discuss the Seebeck coefficient as well as the sheet resistance for application-specific different temperature set points of several doped poly-Si, poly-SiGe, and silicides.
RESUMO
The electrical transport in bismuth nanowires is strongly influenced by both sample geometry and crystallinity. Compared to bulk bismuth, the electrical transport in nanowires is dominated by size effects and influenced by surface states, which gain increasing relevance with increasing surface-to-volume ratios, i.e. with decreasing wire diameter. Bismuth nanowires with tailored diameter and crystallinity constitute, therefore, excellent model systems, allowing to study the interplay of the different transport phenomena. Here, we present temperature-dependent Seebeck coefficient and relative electrical resistance measurements of parallel bismuth nanowire arrays with diameters between 40 and 400 nm synthesized by pulsed electroplating in polymer templates. Both electrical resistance and Seebeck coefficient exhibit a non-monotonic temperature dependence, with the sign of the Seebeck coefficient changing from negative to positive with decreasing temperature. The observed behavior is size-dependent and is attributed to limitations of the mean free path of the charge carriers within the nanowires. The observed size-dependent Seebeck coefficient and in particular the size-dependent sign change opens a promising avenue for single-material thermocouples with p- and n-legs made from nanowires with different diameters.
RESUMO
Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight α-helices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant KD of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146-153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.
Assuntos
Epitopos/imunologia , Mioglobina/imunologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Ressonância de Plasmônio de Superfície/métodos , Animais , Anticorpos Imobilizados/imunologia , Anticorpos Monoclonais/imunologia , Biomarcadores , Epitopos/análise , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Mioglobina/química , Mapeamento de Peptídeos , Ressonância de Plasmônio de Superfície/instrumentaçãoRESUMO
This paper introduces a new methodology for the fabrication of strain-sensor elements for MEMS and NEMS applications based on the tunneling effect in nano-granular metals. The strain-sensor elements are prepared by the maskless lithography technique of focused electron-beam-induced deposition (FEBID) employing the precursor trimethylmethylcyclopentadienyl platinum [MeCpPt(Me)(3)]. We use a cantilever-based deflection technique to determine the sensitivity (gauge factor) of the sensor element. We find that its sensitivity depends on the electrical conductivity and can be continuously tuned, either by the thickness of the deposit or by electron-beam irradiation leading to a distinct maximum in the sensitivity. This maximum finds a theoretical rationale in recent advances in the understanding of electronic charge transport in nano-granular metals.
Assuntos
Nanopartículas Metálicas/química , Nanotecnologia/métodos , Sistemas Microeletromecânicos/métodosRESUMO
Many laser material processing applications require an optimized beam profile, e.g., ring shape or Top-Hat profiles with homogeneous intensity distribution. In this study, we show a beam shaping concept leading to a phase shifting element with binary height profile as well as a very low periodicity with near diffraction limited spot size. Further advantages of so-called Fundamental Beam Mode Shaping (FBS) elements are the simplified handling, and a high efficiency and homogeneity. The calculated height profile of FBS elements are transferred in fused silica substrates using a combination of microlithography technologies, reactive ion etching (RIE) and ion beam etching (IBE). The experiments demonstrated a linear relation between the etching depth after RIE and IBE. The optical evaluation of the manufactured FBS beam mode shaper confirmed the presented concept design.