Особенности акушерско-гинекологического консультирования беременных, страдающих эпилепсией

БЕРЕМЕННОСТИ ОСЛОЖНЕНИЯЭПИЛЕПСИ

A Biotechnology Perspective on Silicon Nanowire FETs for Biosensor Applications

The study of silicon nanowire-FET-based electronic biosensor applications is an emerging scientific field. These biosensors have the benefit of being theoretically extremely sensitive and reports of down to femtomolar (fM) levels of biomolecule detection have been reported. This thesis is written from a biotechnological perspective on the development of a silicon nanowire-FET biosensor. The thesis project was oriented towards developing a novel affinity-based silicon nanowire-FET biosensor based on small (2-3 nm) protein affinity-binders denoted Affibody molecules. The hypothesis was that a smaller biological detector element would reduce the effect of Debye screening of the charged biomarker. This hypothesis was neither proved nor disproved, and a substantial amount of time and effort was spent on improving the function of the different biosensor components. In paper I, a study on the effect of the redox state and pH at solvent-to-surface interfaces of the reference gate electrodes was done by using solutions with alternating pH and varying ratios of the Fe(CN)63-/ Fe(CN)64- redox pair. These experiments showed that the selection of reference gate electrode has major implications on the signal readout in terms of false response and current instability. While a current drop due to potential change on the surface of a platinum reference electrode was observed, no such thing was observed using a silver/silver chloride reference gate electrode. The conclusion is that it is critical for performance to use a reference gate electrode that has a stable electrode potential such as silver/silver chloride. In paper II, a discovery was made when intending to use nanowire joule heating to lyse HT-29 and MCF-7 cells. Using fringing electric fields irreversible electroporation of a cell on top of a nanowire was achieved at 600-1200 mVpeak-to-peak at 10 MHz for 2 ms. The process was monitored using a 3,3´-dihexyloxacarbocyanine iodide (DiOC6(3)) and Propidium Iodide (PI) live-dead dye kit. The nanowire-mediated electroporation method releases the cell content without the risk of heat denaturation and it is ultra-localized. To address the concern on how to control and monitor organosilane monolayer formation in the surface functionalization of silicon nanowires, a microwave-assisted method was evaluated in paper III. Using ellipsometry, AFM, ATR-FTIR and fluorescence scanning it was shown that less than 10 minutes of incubation in 1% (v/v) APTES in toluene at 75⁰C is needed for formation of a 0.7 nm monolayer. In paper IV, surface functionalization was further explored by using microdispensing of solutions of capture probes for localized functionalization of individual devices for a multiplexed silicon nanowire-FET biosensor application. Besides showing by fluorescent scanning that oligonucleotide or protein spots of ~ 100 μm diameters could be deposited on individual silicon nanowires, the functionalization chemistry was validated by using the same protocol for immobilization of the Z domain from Staphylococcus aureus Protein A (SpA) on silicon dioxide-coated SPR sensor chips, followed by real-time detection of the binding of immunoglobulin G. The immunoglobulins as affinity-binders have a drawback due to large size and the importance of having the binding event near the device in silicon nanowire-FET biosensor due to the effect of Debye screening. In paper V, in an effort to further minimize the size of affinity-binders of potential value as capture probes in silicon nanowire-FET applications, a backbone-cyclized, minimized 2-helix affibody-molecule (ZHER2:342min) was designed and produced by Solid Phase Peptide Synthesis(SPPS). The 2-helix affibody-molecule was evaluated for in vivo molecular imaging of HER2-expressing tumours, which was demonstrated in mice carrying SKOV-3 xenografts.QC 20131111</p

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

Introduction Affibody molecules, small scaffold proteins, have demonstrated an appreciable potential as imaging probes. Affibody molecules are composed of three alpha-helices. Helices 1 and 2 are involved in molecular recognition, while helix 3 provides stability. The size of Affibody molecules can be reduced by omitting the third alpha-helix and cross-linking the two remaining, providing a smaller molecule with better extravasation and quicker clearance of unbound tracer. The goal of this study was to develop a novel 2-helix Affibody molecule based on backbone cyclization by native chemical ligation (NCL). Methods The HER2-targeting NCL-cyclized Affibody molecule ZHER2:342min has been designed, synthesized and site-specifically conjugated with a DOTA chelator. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. The binding affinity of DOTA-ZHER2:342min was evaluated in vitro. The targeting properties of 111In- and 68 Ga-DOTA-ZHER2:342min were evaluated in mice bearing SKOV-3 xenografts and compared with the properties of 111In- and 68 Ga-labeled PEP09239, a DOTA-conjugated 2-helix Affibody analogue cyclized by a homocysteine disulfide bridge. Results The dissociation constant (KD) for DOTA-ZHER2:342min binding to HER2 was 18 nM according to SPR measurements. DOTA-ZHER2:342min was labeled with 111In and 68 Ga. Both conjugates demonstrated bi-phasic binding kinetics to HER2-expressing cells, with KD1 in low nanomolar range. Both variants demonstrated specific uptake in HER2-expressing xenografts. Tumor-to-blood ratios at 2 h p.i. were 6.1 ± 1.3 for 111In- DOTA-ZHER2:342min and 4.6 ± 0.7 for 68 Ga-DOTA-ZHER2:342min. However, the uptake of DOTA-ZHER2:342min in lung, liver and spleen was appreciably higher than the uptake of PEP09239-based counterparts. Conclusions Native chemical ligation enables production of a backbone-cyclized HER2-binding 2-helix Affibody molecule (ZHER2:342min) with low nanomolar target affinity and specific tumor uptake.NOTICE: this is the author's version of a work that was accepted for publication in Nuclear Medicine and Biology. Changes resulting from the publishing process, such as editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in:Honarvar H, Jokilaasko N, Andersson K, Malmberg J, Rosik D, Orlova A, Eriksson Karlström A, Tolmachev V, Järver P. Evaluation of Backbone-Cyclized HER2-Binding Two-Helix Affibody Molecule for In Vivo Molecular Imaging. Nucl Med Biol, 40,3, 2013 Apr; :378-86 DOI 10.1016/j.nucmedbio.2012.12.009.http://www.sciencedirect.com/science/article/pii/S0969805112003204</p