Electrochemical Sensing of Interactions between DNA and Charged Macrocycles

In this work, we investigated aggregation of native DNA and thiacalix[4]arene derivative bearing eight terminal amino groups in cone configuration using various redox probes on the glassy carbon electrode. It was shown that sorption transfer of the aggregates on the surface of the electrode covered with carbon black resulted in changes in electrostatic interactions and diffusional permeability of the surface layer. Such changes alter the signals of ferricyanide ion, methylene green and hydroquinone as redox probes to a degree depending on their specific interactions with DNA and own charge. Inclusion of DNA in the surface layer was independently confirmed by scanning electron microscopy, electrochemical impedance spectroscopy and experiments with doxorubicin as a model intercalator. Thermal denaturing of DNA affected the charge separation on the electrode interface and the signals of redox probes. Using hydroquinone, less sensitive to electrostatic interactions, made it possible to determine from 10 pM to 1.0 nM doxorubicin (limit of detection 3 pM) after 10 min incubation. Stabilizers present in the commercial medications did not alter the signal. The DNA sensors developed can find future application in the assessment of the complexes formed by DNA and macrocycles as delivery agents for small chemical species.

How Reliable Is the Electrochemical Readout of MIP Sensors?

Electrochemical methods offer the simple characterization of the synthesis of molecularly imprinted polymers (MIPs) and the readouts of target binding. The binding of electroinactive analytes can be detected indirectly by their modulating effect on the diffusional permeability of a redox marker through thin MIP films. However, this process generates an overall signal, which may include nonspecific interactions with the nonimprinted surface and adsorption at the electrode surface in addition to (specific) binding to the cavities. Redox-active low-molecular-weight targets and metalloproteins enable a more specific direct quantification of their binding to MIPs by measuring the faradaic current. The in situ characterization of enzymes, MIP-based mimics of redox enzymes or enzyme-labeled targets, is based on the indication of an electroactive product. This approach allows the determination of both the activity of the bio(mimetic) catalyst and of the substrate concentration.

Development of Emulsion Gels and Macroporous Hydrogels and their Applications to Metal Adsorption and Enzyme Reaction

Emulsion gels, that is, hydrogels containing randomly distributed oil microdroplets, and macroporous hydrogels with randomly distributed, non-interconnected, sphere-like macropores with several micrometers in diameter were prepared by the emulsion-gelation method [1]. This method involves the synthesis of hydrogels in an oil-in-water (O/W) emulsion by free radical copolymerization of a monomer with a cross-linker, followed by the washing (removal) of the dispersed oil as a pore template (porogen). The observations of oil droplets in an emulsion and internal structure of a macroporous hydrogel demonstrate that the oil droplets act as a pore-template. The pore size and porosity can be adjusted by varying the O/W volume ratios and surfactant amounts [2]. These gels are used as a bulk and have excellent diffusional permeability to a solute and solvent. The emulsion-gelation method can yield potentially intelligent gels in which the macropores function as spaces for reaction, separation and storage. Novel emulsion gel adsorbents, that is, polymeric hydrogels containing randomly distributed microdroplets of an organic extractant (an oil-soluble complexing agent), were developed for metal adsorption [3,4]. The emulsion gel containing an organophosphorus extractant and organosulfur extractant successfully adsorbed In (III) and Pd (II) ions, respectively. Novel macroporous polymeric hydrogels were developed to entrap and immobilize lipase as a model enzyme [5]. The lipase immobilized within the macroporous hydrogel successfully catalyzed the hydrolysis of triacetin in a model enzyme reaction without leakage of lipase or loss of activity during repeated use.

A transmural pressure gradient induces mechanical and biological adaptive responses in endothelial cells

A sudden increase in the transmural pressure gradient across endothelial monolayers reduces hydraulic conductivity ( Lp), a phenomenon known as the sealing effect. To further characterize this endothelial adaptive response, we measured bovine aortic endothelial cell (BAEC) permeability to albumin and 70-kDa dextran, Lp, and the solvent-drag reflection coefficients (σ) during the sealing process. The diffusional permeability coefficients for albumin (1.33 ± 0.18 × 10–6 cm/s) and dextran (0.60 ± 0.16 × 10–6 cm/s) were measured before pressure application. The effective permeabilities (measured when solvent drag contributes to solute transport) of albumin and dextran ( Pealb and Pedex) were measured after the application of a 10 cmH2O pressure gradient; during the first 2 h of pressure application, Pealb, Pedex, and Lp were significantly reduced by 2.0 ± 0.3-, 2.1 ± 0.3-, and 3.7 ± 0.3-fold, respectively. Immunostaining of the tight junction (TJ) protein zonula occludens-1 (ZO-1) was significantly increased at cell-cell contacts after the application of transmural pressure. Cytochalasin D treatment significantly elevated transport but did not inhibit the adaptive response, whereas colchicine treatment had no effect on diffusive permeability but inhibited the adaptive response. Neither cytoskeletal inhibitor altered σ despite significantly elevating both Lp and effective permeability. Our data suggest that BAECs actively adapt to elevated transmural pressure by mobilizing ZO-1 to intercellular junctions via microtubules. A mechanical (passive) component of the sealing effect appears to reduce the size of a small pore system that allows the transport of water but not dextran or albumin. Furthermore, the structures of the TJ determine transport rates but do not define the selectivity of the monolayer to solutes (σ).

Albumin transcytosis in mesothelium

Apparent permeability to albumin ( P alb) was measured with125I-albumin in specimens of rabbit parietal pericardium from lumen to interstitium (L-I) and from interstitium to lumen (I-L). With albumin concentration (Calb) 0.5%, P alb (× 10−5 cm/s) L-I at 37°C was 0.172 ± 0.019 SE; it decreased to 0.092 ± 0.022 I-L at 37°C, 0.089 ± 0.021 L-I at 12°C, and 0.084 ± 0.018 I-L at 12°C. These findings provide evidence for an active transport L-I, likely transcytosis. With Calb 2.5%, 0.05%, and 0.005%, P alb L-I at 37°C was 0.188 ± 0.023, 0.156 ± 0.021, and 0.090 ± 0.021, respectively; at 12°C it was 0.089 ± 0.017, 0.083 ± 0.019, and 0.087 ± 0.026, respectively. Hence, active albumin transport ceases with Calb 0.005%; P alb values I-L at 12°C and with Calb 0.005% are similar and provide diffusional permeability. With physiological Calb (∼1%), active albumin flux was ∼5 × 10−4μmol · h−1 · cm−2. Apparent permeability to FITC-dextran 70 ( P dx) was also measured. P dx (× 10−5 cm/s) L-I at 37°C with Calb 0.5% was 0.095 ± 0.018; it decreased to 0.026 ± 0.004 I-L (37°C, Calb 0.5%), 0.038 ± 0.007 at 12°C (L-I, Calb 0.5%), 0.030 ± 0.009 with Calb 0.005% (L-I, 37°C), and 0.032 ± 0.011 with nocodazole (L-I, 37°C, Calb 0.5%). These findings provide evidence for transcytosis and confirm conclusions drawn from P alb. Vesicular liquid flow, computed from vesicular dextran flux (fluid-phase only), was ∼3.5 μl · h−1 · cm−2. Transcytosis seems a relevant mechanism, removing protein and liquid from serous cavities.

Macromolecule transfer through mesothelium and connective tissue

Diffusional permeability ( P) to inulin ( P in), albumin ( P alb), and dextrans [70 ( P dx 70), 150 ( P dx 150), 550 ( P dx 550), and 2,000 ( P dx 2,000)] was determined in specimens of parietal pericardium of rabbits, which may be obtained with less damage than pleura. P in, P alb, P dx 70, P dx 150, P dx 550, and P dx 2,000 were 0.51 ± 0.06 (SE), 0.18 ± 0.03, 0.097 ± 0.021, 0. 047 ± 0.011, 0.025 ± 0.004, and 0.021 ± 0.005 × 10−5cm/s, respectively. P in, P alb, and P dx 70 of connective tissue, obtained after removal of mesothelium from specimens, were 10.3 ± 1.42, 2.97 ± 0.38, and 2.31 ± 0.16 × 10−5 cm/s, respectively. Hence, P in, P alb, and P dx 70 of mesothelium were 0.54, 0.20, and 0.10 × 10−5 cm/s, respectively. Inulin (like small solutes) fitted the relationship P-solute radius for restricted diffusion with a 6-nm “pore” radius, whereas macromolecules were much above it. Hence, macromolecule transfer mainly occurs through “large pores” and/or transcytosis. In line with this, the addition of phospholipids on the luminal side (which decreases pore radius to ∼1.5 nm) halved P in but did not change P alband P dx 70. P in is roughly similar in mesothelium and capillary endothelium, whereas P to macromolecules is greater in mesothelium. The albumin diffusion coefficient through connective tissue was 17% of that in water. Mesothelium provides 92% of resistance to albumin diffusion through the pericardium.

Equivalent radius of paracellular “pores” of the mesothelium

Diffusional permeability ( P) to water ( P w), Cl−([Formula: see text]), and mannitol ( P man) was determined in specimens of rabbit parietal pericardium without and with phospholipids added on the luminal side, as previously done with sucrose and Na+. P to the above-mentioned molecules and to Na+([Formula: see text]) was also determined after mesothelium was scraped away from specimens. P w,[Formula: see text],[Formula: see text], and P man of connective tissue were the following (×10−5 cm/s): 73.1 ± 7.3 (SE), 59.5 ± 4.5, 41.7 ± 3.4, and 23.4 ± 2.4, respectively. From these and corresponding data on integer pericardium, P w,[Formula: see text],[Formula: see text], and P man of mesothelium were computed. They were the following: 206, 17.9, 9.52, and 3.93, and 90.2, 14.4, 4.34, and 1.75 × 10−5cm/s without and with phospholipids, respectively. As previously found for P to sucrose, P to solutes is smaller in mesothelium than in connective tissue, although the latter is ∼35-fold thicker; instead, P w is higher in mesothelium, suggesting marked water diffusion through cell membrane. Equivalent radius of paracellular “pores” of mesothelium was computed with two approaches, disregarding P w. The former, a graphical analysis on a P-molecular radius diagram, yielded 6.0 and 1.7 nm without and with phospholipids, respectively. The latter, on the basis of P man, P to sucrose, and function for restricted diffusion, yielded 7.8 and 1.1 nm, respectively.