Bilayer reconstitution of voltage-dependent ion channels using a microfabricated silicon chip

Painted bilayers containing reconstituted ion channels serve as a well defined model system for electrophysiological investigations of channel structure and function. Horizontally oriented bilayers with easy solution access to both sides were obtained by painting a phospholipid:decane mixture across a cylindrical pore etched into a 200-microm thick silicon wafer. Silanization of the SiO(2) layer produced a hydrophobic surface that promoted the adhesion of the lipid mixture. Standard lithographic techniques and anisotropic deep-reactive ion etching were used to create pores with diameters from 50 to 200 microm. The cylindrical structure of the pore in the partition and the surface treatment resulted in stable bilayers. These were used to reconstitute Maxi K channels in the 100- and 200-microm diameter pores. The electrophysiological characteristics of bilayers suspended in microchips were comparable with that of other bilayer preparations. The horizontal orientation and good voltage clamping properties make the microchip bilayer method an excellent system to study the electrical properties of reconstituted membrane proteins simultaneously with optical probes.     

Dependence of adhesive behavior of neutrophils on local fluid dynamics in a region with recirculating flow

We have recently described patterns of adhesion of different types of leukocytes downstream of a backward facing step. Here the predicted fluid dynamics in channels incorporating backward facing steps are described, and related to the measured velocities of flowing cells, patterns of attachment and characteristics of rolling adhesion for neutrophils perfused over P-selectin. Deeper (upstream depth 300 microm, downstream depth 600 microm, maximum wall shear stress approximately 0.1 Pa) and shallower (upstream depth 260 microm, downstream depth 450 microm, maximum wall shear stress approximately 0.3 Pa) channels were compared. Computational fluid dynamics (CFD) predicted the presence of vortices downstream of the steps, distances to reattachment of flow, local wall shear stresses and components of velocity parallel and perpendicular to the wall. Measurements of velocities of perfused neutrophils agreed well with predictions, and suggested that adhesion to P-selectin should be possible in the regions of recirculating flow, but not downstream in re-established flow in the high shear channel. When channels were coated with a P-selectin-Fc chimaera, neutrophils were captured from flow and immobilised. Capture showed local maxima around the reattachment points, but was absent elsewhere in the high shear chamber. In the low shear chamber there was depression of adhesion just beyond the reattachment point because of expansion of flow and depletion of neutrophils near the wall. Inside the recirculation zones, adhesion decreased approaching the step because of an increasing, vertically upward velocity component. When channels were coated with P-selectin, neutrophils rolled in all regions, but lifted off the surface as they rolled backwards into low shear regions near the step. Rolling velocity in the recirculation zone was independent of shear stress, possibly because of the effects of vertical lift. We conclude that while local wall shear stress influences adhesive behavior, delivery of cells to the wall and their behavior after capture also depend on components of flow perpendicular to the wall.     

Fabrication of lab-on chip platforms by hot embossing and photo patterning

In this paper, we review the approaches developed in our laboratory to fabricate polymer-based microfluidic devices to suit a range of applications in bio- or chemical analysis. Thermoplastic materials such as polycarbonate (PC) and poly(methyl methacrylate) (PMMA) are used to fabricate microfluidic devices via hot embossing. To emboss microchannels, we use hard stamps fabricated in silicon or soft stamps molded on poly(dimethylsiloxane) (PDMS). Hard stamps are fabricated on silicon wafers through photolithography and deep reactive ion etching (DRIE). Soft stamps are fabricated by casting PDMS prepolymer on silicon molds. To enclose the fluidic channels, direct fusion bonding was found to produce the highest bond strength with minimal structural deformation. One-step photolithographic methods have also been explored to produce via photochemical patterning microfluidic structures in photocurable materials. We use the photocurable capabilities of a PDMS copolymer, which incorporates a methacrylate crosslinker. Microfluidic channels are produced via one step-photopatterning processes by crosslinking the prepolymer mixture through a photomask. The smaller feature size attainable was 100 microm. Structures with higher spatial resolution are fabricated through a photoimprinting process whereby a mold is pressed against the precured mixture during UV crosslinking exposure. The application of the fabricated fluidic devices in electrophoretic ion analysis is also presented.     

Serially etched shark enameloid observed by incident light microscopy

Longitudinal and transverse tooth sections of Isurus oxyrinchus were serially etched in 2.6% nitric acid. The changing optical properties of the etched surfaces were observed during the serial etchings, and the descent of the enameloid surfaces was measured. Shark enameloid seems to be less effectively etched by acid than human enamel; this difference may be due to differences in solubility between fluorapatite and hydroxyapatite. Most of the information regarding the structure of the enameloid was gained during the first five of ten etchings. The reflection of light from the surface was influenced by the orientation of the crystallites, longitudinally sectioned crystallites reflecting the light better than transversely sectioned crystallites. The dentinal extensions were continuous with and of the same structure as the underlying dentine. The radial fibers originated from the dentinal extensions, and they both contained organic material and were accompanied by crystallites. When the specimens were imbibed with water the distinctness of the dentinal extensions and radial fibers was improved.     

Specific layers in aerobically grown microbial granules

AIMS: To determine the optimal size of aerobically grown granules for wastewater treatment by measuring specific layers within the granules. METHODS AND RESULTS: A variety of biological layers were detected by oligonucleotide probes, specific fluorochromes, and fluorescent microspheres. The channels in the granule matrix penetrated to depths of 900 microm. A layer of obligate anaerobic bacteria was detected at a depth of 800 microm below the granule surface. Dead cells were also observed in the granule interior. CONCLUSIONS: Aerobically grown granules contained layers of aerobic and anaerobic micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimal diameter of the aerobic granule is less than 1600 microm. This is twice the distance from the granule surface to the anaerobic layer. This approach can be used to optimize the thickness of other microbial aggregates such as flocs, colonies and biofilms.     

Impact of nano-topography on bacterial attachment

The adhesion of bacteria to surfaces is an important biological process, but one that has resisted simple categorization due to the number and complexity of parameters involved. The roughness of the substrate is known to play a significant role in the attachment process, particularly when the surface irregularities are comparable to the size of the bacteria and can provide shelter from unfavorable environmental factors. According to this scenario, roughness on a scale much smaller than the bacteria would not be expected to influence the initial attachment. To test this hypothesis, the impact of nanometer-scale roughness on bacterial attachment has been investigated using as-received and chemically etched glass surfaces. The surface modification by etching resulted in a 70% reduction in the nanoscale roughness of the glass surface with no significant alteration of its chemical composition or charge. Nevertheless, the number of bacteria adhering to the etched surface was observed to increase by a factor of three. The increase in attachment was also associated with an alteration in cellular metabolic activity as demonstrated by changes in characteristic cell morphologies and increased production of extracellular polymeric substances. The results indicate that bacteria may be more sensitive to nanoscale surface roughness than was previously believed.     

Er,Cr:YSGG激光切割对牙体与充填物粘结力影响的研究

本文用 1千牛电液疲劳实验机检测充填材料与牙面之间的粘结力 ,发现 :在牙釉质组中 ,酸蚀组、6瓦激光切割组及 6瓦激光切割联合酸蚀组抗剪切粘结强度无显著性差异 (P >0 .0 5) ;在牙本质组中 ,酸蚀组、4瓦激光切割组及 4瓦激光切割联合酸蚀组抗剪切粘结强度也无显著性差异 (P >0 .0 5)。并通过SEM观察激光处理后牙体表面结构的变化 ,发现激光切割后牙釉质表面不平呈层状 ,无玷污层 ,釉柱未见破坏 ;牙本质小管开放 ,表面无玷污层 ,达到酸蚀的效果。因此Er,Cr :YSGG激光切割牙体硬组织具有传统钻切割与酸蚀刻的联合作用 ,可以代替传统的酸蚀方法。     

Modeling of discrete currents of single ion channels of cell membranes using synthetic nanometer pores in polyethylene terephthalate films

In studying of conductivity of single supernarrow pores (varying 1 to 15 nm in diameter), formed in thin membranes (10-12 microm in the thickness) from polyethylene terephthalate (PETP), there were revealed discrete changes of currents passing through such pores when applied from external source of potential difference from 200 to 1000 mV. By several characteristics, such discrete currents (discrete conductivity changes) appeared to be identical the so-cold current of single ionic channels in the cell membranes. Supernarrow pores which properties are describes in the present work were obtained as a result of alkaline etching of tracks in thin PETP membranes (a variant of the so-called nuclear filters). Alkaline etching leads to formation of negative fixed charges on the walls of the pores compensated by positive counterions. When setting potential difference onto the PETP membrane, the latter cation layer is able to transfer the current and this transfer was called the surface conductance. In the case of nanometer pores, such surface conductance may be dominating. We have shown that these discrete changes of currents passing through nanometer pores are associated with metastability of the surface conductance. In the case of highly cation-selective channels in the cell membranes it is inevitable, that at least a part of these channels should have dominating cation surface conductance and mentioned above conductance metastability as well. Our findings allow us to propose a new explanation of the origin of the characteristic discreteness of the currents of cation-selective ionic channels in the cell membranes.     

Oriented Schwann cell growth on microgrooved surfaces

Silicon wafers bearing microgrooved surfaces with various groove width, spacing, and depth were fabricated using microlithography. The orientation of rat Schwann cells along the direction of the grooves was measured at 24 h after seeding the cells. When the width/spacing of the grooves was fixed at 10/10 microm, the mean percentage of aligned cells was 12% for grooves of 0.5 microm depth, 15% for those of 1 microm depth, and 26% for those of 1.5 microm depth (P < 0.05). When the depth of grooves was fixed at 1.5 microm, the mean percentage of aligned cells increased from 26% for width/spacing 10/10 microm, to 33% for 10/20 microm or 20/10 microm, and up to 41% for 20/20 microm (P < 0.05). On the surface with grooves of width/spacing/depth = 20/20/1.5 microm and modified by laminin, the alignment at 24 h approached 60%, versus 51% for collagen-coated surface and 41% for uncoated surface (P < 0.05). At 48 h after seeding, about 66% of the cells were aligned on the above laminin-modified surface. The groove depth influenced orientation of Schwann cells significantly. The cell alignment on 20/20/3 microm microgrooved poly(D,L-lactide-co-glycolide) 90:10 (PLGA) surfaces transferred from silicon reached 72% at 48 h and 92% at 72 h (P < 0.05). Coating this surface with laminin enhanced cell alignment only in short term (67% vs. 62% at 24 h, P < 0.05). The cell alignment guided by surface microgrooves was time dependent.     

Micropillar array chip for integrated white blood cell isolation and PCR

We report the fabrication of silicon chips containing a row of 667 pillars, 10 by 20 microm in cross-section, etched to a depth of 80 microm with adjacent pillars being separated by 3.5 microm. The chips were used to separate white blood cells from whole blood in less than 2 min and for subsequent PCR of a genomic target (eNOS). Chip fluid dynamics were validated experimentally using CoventorWare microfluidic simulation software. The amplicon concentrations were determined using microchip capillary electrophoresis and were >40% of that observed in conventional PCR tubes for chips with and without pillars. Reproducible on-chip PCR was achieved using white blood cell preparations isolated from whole human blood pumped through the chip.     

Ion beam treatment of titanium surfaces for enhancing deposition of hydroxyapatite from solution

Surface coating with hydroxyapatite (HA) is a common way to improve the osseointegration of orthopaedic and dental titanium (Ti)-based materials. The main problems with current techniques are changes in composition during heating and poor adhesion to the surface. An alternative method is deposition of HA onto an activated surface out of a solution. The present work studies the surface treatment involving ion implantation of Na into Ti to induce a modification in chemistry and morphology, showing sodium titanate (Na(2)TiO(3)) incorporated within the surface layer with concentration, depth distribution, and morphology depending on the parameters of the ion implantation. Such ion-implanted Ti surfaces actively induce heterogeneous precipitation of HA from a simulated body fluid containing physiological concentrations of calcium and phosphate ions. This is compared with the activation by NaOH etching. The growth of bone forming cells on the pure Na implanted surface is oriented without an increased bone formation. Cell growth on the NaOH etched surface is reduced. After deposition of HA on both surfaces cell the growth pattern was improved.     

Multiphoton excitation fluorescence microscopy and spectroscopy of in vivo human skin

Multiphoton excitation microscopy at 730 nm and 960 nm was used to image in vivo human skin autofluorescence from the surface to a depth of approximately 200 microm. The emission spectra and fluorescence lifetime images were obtained at selected locations near the surface (0-50 microm) and at deeper depths (100-150 microm) for both excitation wavelengths. Cell borders and cell nuclei were the prominent structures observed. The spectroscopic data suggest that reduced pyridine nucleotides, NAD(P)H, are the primary source of the skin autofluorescence at 730 nm excitation. With 960 nm excitation, a two-photon fluorescence emission at 520 nm indicates the presence of a variable, position-dependent intensity component of flavoprotein. A second fluorescence emission component, which starts at 425 nm, is observed with 960-nm excitation. Such fluorescence emission at wavelengths less than half the excitation wavelength suggests an excitation process involving three or more photons. This conjecture is further confirmed by the observation of the super-quadratic dependence of the fluorescence intensity on the excitation power. Further work is required to spectroscopically identify these emitting species. This study demonstrates the use of multiphoton excitation microscopy for functional imaging of the metabolic states of in vivo human skin cells.     

Dual-wavelength phosphorimetry for determination of cortical and subcortical microvascular oxygenation in rat kidney.

This study presents a dual-wavelength phosphorimeter developed to measure microvascular PO2 (microPO2) in different depths in tissue and demonstrates its use in rat kidney. The used phosphorescent dye is Oxyphor G2 with excitation bands at 440 and 632 nm. The broad spectral gap between the excitation bands combined with a relatively low light absorption of 632 nm light by tissue results in a marked difference in penetration depths of both excitation wavelengths. In rat kidney, we determine the catchments depth of the 440-nm excitation to be 700 microm, whereas the catchments depth of 632 nm is as much as 4 mm. Therefore, the measurements differentiate between cortex and outer medulla, respectively. In vitro, no difference in PO2 readings between both channels was found. On the rat kidney in vivo, the measured cortical microPO2 was on average 20 Torr higher than the medullary microPO2 over a wide PO2 range induced by variations in inspired oxygen fraction. Examples provided from endotoxemia and resuscitation show differences in responses of mean cortical and medullary PO2 readings as well as in the shape of the PO2 histograms. It can be concluded that oxygen-dependent quenching of phosphorescence of Oxyphor G2 allows quantitative measurement of microPO2 noninvasively in two different depths in vivo. Oxygen levels measured by this technique in the rat renal cortex and outer medulla are consistent with previously published values detected by Clark-type oxygen electrodes. Dual-wavelength phosphorimetry is excellently suited for monitoring microPO2 changes in two different anatomical layers under pathophysiological conditions with the characteristics of providing oxygen histograms from two depths and having a penetration depth of several millimeters.     

Nucleic acid purification using microfabricated silicon structures

A microfluidic device has been designed, fabricated and tested for its ability to purify bacteriophage lambda DNA and bacterial chromosomal DNA, a necessary prerequisite for its incorporation into a biosensor. This device consists of a microfabricated channel in which silica-coated pillars were etched to increase the surface area within the channel by 300-600%, when the etch depth is varied from 20 to 50 microm. DNA was selectively bound to these pillars in the presence of the chaotropic salt guanidinium isothiocyanate, followed by washing with ethanol and elution with low-ionic strength buffer. Positive pressure was used to move solutions through the device, removing the need for centrifugation steps. The binding capacity for DNA in the device was approximately 82 ng/cm2 and on average, 10% of the bound DNA could be purified and recovered in the first 50 microl of elution buffer. Additionally, the device removed approximately 87% of the protein from a cell lysate. Nucleic acids recovered from the device were efficiently amplified by the polymerase chain reaction suggesting the utility of these components in an integrated, DNA amplification-based biosensor. The miniaturized format of this purification device, along with its excellent purification characteristics make it an ideal component for nucleic acid-based biosensors, especially those in which nucleic acid amplification is a critical step.     

The effects of surface configuration on the retentive properties of parallel-sided dowels cemented with a composite luting agent

Factors which have effect on the retention of any dowel include the shape, length, diameter, and surface configuration of the dowel, the cementing medium, precision fitness and material, etc. The shape and surface configuration are the most important among all factors. Recent developments of the etching technique for cast metal has improved the bonding strength between base metal alloys and composite cement which offers a great opportunity for better retention of the dowel if its surface has been etched. In the past, because it has often been ignored that the bonding at the cement-dentin interface is weaker than that between the metal and cement, the true impact of surface configuration and cement thickness on the retention of dowels has never really been observed. In this study, plastic transparent rods were substituted for natural teeth to avoid anatomical variation and circular horizontal grooves were placed on the channel walls to strengthen the retention between the cement and the plastic rods. A total of 140 dowel samples were cast in Rexillium III and were divided into seven groups. The surfaces of the samples were treated differently by smoothing, sandblasting, chemical etching, electrolytic etching and serrating, and fixed into the channels of the plastic rods by cementation with composite resin. Immediately thereafter, the samples were submerged in 37 degrees C water for 7 days. Instron crosshead speed 1 mm/min was used in measuring the tensile forces required for extracting the dowels from the channels. Data were analyzed with the ANOVA method. Among the three factors known to have an effect on the retention being considered in this study, the circular horizontal groove proved to be the most influential, and surface configuration came next, while cement thickness was the least influential. Among the different surface configurations, the serrated dowels showed the best retention and were followed in order by etched and smooth or sandblasted dowels.     

The power of single and multibeam two-photon microscopy for high-resolution and high-speed deep tissue and intravital imaging

Two-photon microscopy is indispensable for deep tissue and intravital imaging. However, current technology based on single-beam point scanning has reached sensitivity and speed limits because higher performance requires higher laser power leading to sample degradation. We utilize a multifocal scanhead splitting a laser beam into a line of 64 foci, allowing sample illumination in real time at full laser power. This technology requires charge-coupled device field detection in contrast to conventional detection by photomultipliers. A comparison of the optical performance of both setups shows functional equivalence in every measurable parameter down to penetration depths of 200 microm, where most actual experiments are executed. The advantage of photomultiplier detection materializes at imaging depths >300 microm because of their better signal/noise ratio, whereas only charge-coupled devices allow real-time detection of rapid processes (here blood flow). We also find that the point-spread function of both devices strongly depends on tissue constitution and penetration depth. However, employment of a depth-corrected point-spread function allows three-dimensional deconvolution of deep-tissue data up to an image quality resembling surface detection.     

Immuno-capture of Cryptosporidium parvum using micro-well array

A glass slide and micro-well array chip on which anti-Cryptosporidium parvum antibody was immobilized were used for the rapid capture and detection of C. parvum. Biotinylated anti-C. parvum antibodies were spotted onto the streptavidin-coated glass slides. C. parvum oocysts were captured specifically on the spot when more than 73 ng of anti-C. parvum antibody was applied onto the glass slide. However, C. parvum oocysts captured on the glass slide were detached by repeating washing steps. To improve the capture efficiency of oocysts, capture was performed in a micro-well format consisting of 1024 wells/2.5 mm2 (32 x 32 wells) fabricated as a chip by photolithography. Instead of a flat surface on a glass slide, each well was 30 microm in diameter and 10 microm in depth. Streptavidin was also immobilized onto the micro-well array. The biotinylated anti-C. parvum antibodies were immobilized efficiently onto the chip using a buffer containing 20% methanol. Using this technique C. parvum oocysts were stably captured onto the chip after repeated washing procedures. These data show that the newly designed micro-well array technique described here is useful for antibody-mediated C. parvum capture.     

Depth-varying density and organization of chondrocytes in immature and mature bovine articular cartilage assessed by 3d imaging and analysis.

Articular cartilage is a heterogeneous tissue, with cell density and organization varying with depth from the surface. The objectives of the present study were to establish a method for localizing individual cells in three-dimensional (3D) images of cartilage and quantifying depth-associated variation in cellularity and cell organization at different stages of growth. Accuracy of nucleus localization was high, with 99% sensitivity relative to manual localization. Cellularity (million cells per cm3) decreased from 290, 310, and 150 near the articular surface in fetal, calf, and adult samples, respectively, to 120, 110, and 50 at a depth of 1.0 mm. The distance/angle to the nearest neighboring cell was 7.9 microm/31 degrees , 7.1 microm/31 degrees , and 9.1 microm/31 degrees for cells at the articular surface of fetal, calf, and adult samples, respectively, and increased/decreased to 11.6 microm/31 degrees , 12.0 microm/30 degrees , and 19.2 microm/25 degrees at a depth of 0.7 mm. The methodologies described here may be useful for analyzing the 3D cellular organization of cartilage during growth, maturation, aging, degeneration, and regeneration.     

William Hogarth,Unwitting Neurochemist?

William Hogarth's famous etching Gin Lane is often used to illustrate the debilitating results of alcohol addiction. Less well known is the companion etching Beer Street in which death, murder and squalor are replaced by health, orderliness and joy. Some 250 years later, the rise of science, and specifically of neurochemical research, has defined how the malnutrition, including avitaminosis, resulting from addiction to distilled spirits (rather than more judicious use of less potent alcoholic beverages) disturbs brain metabolism and function. These two etchings, which have survived for their historical and artistic value, continue to have sociological and clinical relevance.     

Distribution of Clostridium perfringens in polluted lake environments

Many of the directives that relate to the prevention of pollution or the improvement of fresh water also relate to lake waters since lake waters ultimately inherit much of the pollution that enters into fresh water. In order to determine the influence of the water depth on Clostridium perfringens, we utilised a new medium, lactose-sulfite (LS) broth, suggested for rapid enumeration and identification of C. perfringens. Duplicate samples were collected at each one of the following sites of the polluted station: surface, 60 cm, 90 cm and bottom (1.18 cm). Membrane filtration equipment was used. All samples were alternatively passed through two membrane filters, the first (20-25 microm pore size) was used for retention of the abundant phytoplankton and the second (porosity 0.45 microm) for C. perfringens. Membranes were placed into the first tube of ten-fold dilutions from 10(1) to 10(4) and incubated aerobically in a waterbath at 46 degrees C for 24 h. The numbers of C. perfringens fluctuated depending on the water depth. Vegetative forms were found only in the bottom sampling; they were never found in surface, 60 cm and 90 cm sampling sites. Sporulated forms were found in all sampling sites with the exception of the surface sampling. Clostridium perfringens as an anaerobic bacterium never occurred in the surface waters in vegetative or spore forms, even if the waters were extremely polluted by domestic or industrial activities. Vegetative forms occurred only in the bottom samples but spore forms which are more resistant to various environmental effects occurred in all depths except for the surface.