3D porous structure imaging of membranes for medical devices using scanning probe microscopy and electron microscopy: from membrane science points of view.

The evolution of hemodialysis membranes (dialyzer, artificial kidney) was remarkable, since Dow Chemical began manufacturing hollow fiber hemodialyzers in 1968, especially because it involved industrial chemistry, including polymer synthesis and membrane manufacturing process. The development of hemodialysis membranes has brought about the field of medical devices as a major industry. In addition to conventional electron microscopy, scanning probe microscopy (SPM), represented by atomic force microscopy (AFM), has been used in membrane science research on porous membranes for hemodialysis, and membrane science contributes greatly to the hemodialyzer industry. Practical studies of membrane porous structure-function relationship have evolved, and methods for analyzing membrane cross-sectional morphology were developed, such as the ion milling method, which was capable of cutting membrane cross sections on the order of molecular size to obtain smooth surface structures. Recently, following the global pandemic of SARS-CoV-2 infection, many studies on new membranes for extracorporeal membrane oxygenator have been promptly reported, which also utilize membrane science researches. Membrane science is playing a prominent role in membrane-based technologies such as separation and fabrication, for hemodialysis, membrane oxygenator, lithium ion battery separators, lithium recycling, and seawater desalination. These practical studies contribute to the global medical devices industry.

Application of mathematical analysis on dialysis.

Hemodialysis is a blood purification method based on solute removal by diffusion and incorporates filtration to improve the efficiency of water removal and removal of high molecular weight substances. It is now a well-established treatment, due to the improved performance of dialyzers. This review outlines the development process of dialyzers, focusing on the application based on the mathematical analysis. First, phenomena occurring in the vicinity of the dialysis membrane are explained using a film model for diffusion and a gel polarization model for filtration. Then, currently established dialyzer designs are introduced using mathematical analysis. Furthermore, the design of dialyzers to promote internal filtration, the designs of hemodiafilters suitable for online hemodiafiltration (HDF), and the design of compact dialyzer for are also presented.

Impact of three-dimensional tortuous pore structure on polyethersulfone membrane morphology and mass transfer properties from a manufacturing perspective.

We examined typical commercial poly(ethersulfone) (PESf) hemodialysis and hemoconcentration membranes successfully used in manufacturing, and employed scanning probe microscope (SPM) to achieve a structural observation of the pores in the inner membrane surfaces, as well as measure the pore diameters and their distribution, verifying the relationship between the typical mass transfer properties. We focused on the differences between the PESf membranes which were expected to further improve the advanced pore structure control and functional design for various medical uses. The three-dimensional tortuous capillary pores on the inner surface of hollow fiber hemodialysis and hemoconcentrator membranes were investigated using dynamic force microscopy (DFM), and the pore diameter and distribution were measured through a line analysis. Compared with PUREMA-A, PES-Sα hemodialysis membranes have smaller three-dimensional tortuous capillary pore diameters and pore areas, as well as a smaller pore diameter distribution and pore area distribution, which make the accurate measurements of the pore diameter using FE-SEM impossible. These PESf membranes are almost the same in pure water permeability, but greatly differ in pore diameter and pore diameter distribution. By comparing and verifying as above, we may gain insight into the flexibility, versatility, and superior structural and functional controllability of PESf membrane pore structures, which could advance the development of pore structure control. Pending issues include the fact that, using a line analysis software of SPM devices, it is very difficult to measure hundred pores which clearly reflects the poor quality of pore size distributions obtained in this study, measurement accuracy must be improved further.

Effect of ruminal administration of soy sauce oil on rumen fermentation, milk production and blood parameters in dairy cows.

OBJECTIVE: To evaluate soy sauce oil (a by-product of making whole soybean soy sauce) as a new dietary lipid source, a large amount of soy sauce oil was administered into the rumen of dairy cows. METHODS: Four Holstein dairy cows fitted with rumen cannulae were used in a 56-day experiment. Ruminal administration of soy sauce oil (1 kg/d) was carried out for 42 days from day 8 to day 49 to monitor nutritional, physiological and production responses. RESULTS: Dry matter intake and milk yield were not affected by soy sauce oil administration, whereas 4% fat-corrected milk yield and the percentage of milk fat decreased. Although ruminal concentration of total volatile fatty acids (VFA) and the proportion of individual VFA were partially affected by administration of soy sauce oil, values were within normal ranges, showing no apparent inhibition in rumen fermentation. Administration of soy sauce oil decreased the proportions of milk fatty acids with a carbon chain length of less than 18, and increased the proportions of stearic, oleic, vaccenic and conjugated linoleic acids. Conjugated linoleic acid content in milk became 5.9 to 8.8 times higher with soy sauce oil administration. Blood serum concentrations of non-esterified fatty acid, 3-hydroxybutyric acid, total cholesterol, free cholesterol, esterified cholesterol, triglyceride and phospholipid increased with administration of soy sauce oil, suggesting a higher energy status of the experimental cows. CONCLUSION: The results suggest that soy sauce oil could be a useful supplement to potentially improve milk functionality without adverse effects on ruminal fermentation and animal health. More detailed analysis is necessary to optimize the supplementation level of this new lipid source in feeding trials.

Technical characterization of dialysis fluid flow and mass transfer rate in dialyzers with various filtration coefficients using dimensionless correlation equation.

The objective of the present study is to evaluate the effect of filtration coefficient and internal filtration on dialysis fluid flow and mass transfer coefficient in dialyzers using dimensionless mass transfer correlation equations. Aqueous solution of vitamin B12 clearances were obtained for REXEED-15L as a low flux dialyzer, and APS-15EA and APS-15UA as high flux dialyzers. All the other design specifications were identical for these dialyzers except for filtration coefficient. The overall mass transfer coefficient was calculated, moreover, the exponents of Reynolds number (Re) and film mass transfer coefficient of the dialysis-side fluid (k D) for each flow rate were derived from the Wilson plot and dimensionless correlation equation. The exponents of Re were 0.4 for the low flux dialyzer whereas 0.5 for the high flux dialyzers. Dialysis fluid of the low flux dialyzer was close to laminar flow because of its low filtration coefficient. On the other hand, dialysis fluid of the high flux dialyzers was assumed to be orthogonal flow. Higher filtration coefficient was associated with higher k D influenced by mass transfer rate through diffusion and internal filtration. Higher filtration coefficient of dialyzers and internal filtration affect orthogonal flow of dialysis fluid.

Experimental evaluation of flow and dialysis performance of hollow-fiber dialyzers with different packing densities.

The dialyzer housing structure should be designed in such a way that high dialysis performance is achieved. To achieve high dialysis performance, the flow of the dialysis fluid and blood should be uniform, without channeling and dead spaces. The objective of this study was to evaluate the effect of fiber packing density on the flow of dialysis fluid and blood, and on the dialysis performance of a hollow-fiber dialyzer at defined flow rates for blood (Q (B) = 200 mL/min), dialysis fluid (Q (D) = 500 mL/min), and filtrate (Q (F) = 0 mL/min). We measured Q (D), Q (B), and solute clearance for 3 test dialyzers with dialyzer housing different diameters. To evaluate the flow of dialysis fluid and blood, we measured the residence time of the dialysis fluid and blood in the test dialyzers by use of the pulse-response method. We also measured the clearances of urea, creatinine, vitamin B(12), and lysozyme to evaluate the dialysis performance of the test dialyzers. At packing densities ranging from 48 to 67%, higher packing densities and lower housing diameters of the dialyzer resulted in higher dialysis performance because the dialysis fluid and blood entered the hollow-fiber bundle smoothly and, hence, increased contact area between the dialysis fluid and the blood led to better dialysis performance.

Poly(N-vinyl-2-pyrrolidone) elution from polysulfone dialysis membranes by varying solvent and wall shear stress.

Some dialysis patients are treated with post-hemodiafiltration (HDF); the blood viscosity of the patients who undergo post-HDF is higher than that of the patients who undergo conventional hemodialysis. This study aims to evaluate poly(N-vinyl-2-pyrrolidone) (PVP) elution from PSf dialysis membranes by varying solvents and high wall shear stress caused by blood viscosity. We tested three commercial membranes: APS-15SA (Asahi Kasei Kuraray), CX-1.6U (Toray) and FX140 (Fresenius). Dialysate and blood sides of the dialyzers were primed with reverse osmosis (RO) water and saline. RO water, saline and dextran solution (2.9 and 5.8 mPa s) were circulated in the blood side. The amount of eluted PVP was determined by 0.02 N iodometry. The hardness and adsorption force of human serum albumin (HSA) on the membrane surfaces were measured by the atomic force microscope. When wall shear stress was increased using dextran, the amount of PVP eluted by the 2.9 mPa s solution equaled that eluted by the 5.8 mPa s solution with APS-15SA and CX-1.6U sterilized by gamma rays. The amount of PVP eluted by the 5.8 mPa s solution was higher than that eluted by the 2.9 mPa s solution with FX140 sterilized by autoclaving. The wall shear stress increased the PVP elution from the surface, hardness and adsorption force of HSA. Sufficient gamma-ray irradiation is effective in decreasing PVP elution.

Insights into Gradient and Anisotropic Pore Structures of Capiox® Gas Exchange Membranes for ECMO: Theoretically Verifying SARS-CoV-2 Permeability.

When using the extracorporeal capillary membrane oxygenator (sample A) for ECMO treatments of COVID-19 severely ill patients, which is dominantly used in Japan and worldwide, there is a concern about the risk of SARS-CoV-2 scattering from the gas outlet port of the membrane oxygenator. Terumo has launched two types of membranes (sample A and sample B), both of which are produced by the microphase separation processes using polymethylpentene (PMP) and polypropylene (PP), respectively. However, the pore structures of these membranes and the SARS-CoV-2 permeability through the membrane wall have not been clarified. In this study, we analyzed the pore structures of these gas exchange membranes using our previous approach and verified the SARS-CoV-2 permeation through the membrane wall. Both have the unique gradient and anisotropic pore structure which gradually become denser from the inside to the outside of the membrane wall, and the inner and outer surfaces of the membrane have completely different pore structures. The pore structure of sample A is also completely different from the other membrane made by the melt-extruded stretch process. From this, the pore structure of the ECMO membrane is controlled by designing various membrane-forming processes using the appropriate materials. In sample A, water vapor permeates through the coating layer on the outer surface, but no pores that allow SARS-CoV-2 to penetrate are observed. Therefore, it is unlikely that SARS-CoV-2 permeates through the membrane wall and scatter from sample A, raising the possibility of secondary ECMO infection. These results provide new insights into the evolution of a next-generation ECMO membrane.

Longer storage of dialyzers increases elution of poly(N-vinyl-2-pyrrolidone) from polysulfone-group dialysis membranes.

The objective of this study was to evaluate the effect of protracted storage of dialyzers on the amount of poly(N-vinyl-2-pyrrolidone) (PVP) eluted from polysulfone-group dialysis membranes. We tested five dialysis membranes: APS-15SA (Asahi Kasei Kuraray, wet), CX-1.6U (Toray, moist), FX140 (Fresenius, dry), PES-15Sα (Nipro, dry), and FDX-150GW (Nikkiso, wet). Each dialyzer was stored for 1, 3, 14, and 18 months after sterilization. The dialysis-fluid side compartment was primed with reverse osmosis (RO) water at 500 mL/min for 5 min at 310 K. The blood side compartment was primed with RO water at 200 mL/min for 5 min at 310 K. Finally, 1 L RO water was circulated through the blood side compartment at 200 mL/min for 4 h at 310 K. Eluted PVP was determined by use of the iodine method, using 0.02 N: iodine solution. PVP was mainly eluted from wet-type dialyzers during priming. Thus, the standard 5 min priming of the wet-type dialyzer according to the maker manual inhibits PVP elution during circulation. PVP was eluted in the dialysis-fluid side of the moist-type dialyzer during priming but no PVP was eluted in the blood side. PVP was mainly eluted from dry-type dialyzers during circulation. We recommend more than the standard 5 min priming, particularly for dry-type dialyzers stored for protracted periods, because 5 min insufficient to inhibit PVP elution during circulation.

Electron Microscopic Confirmation of Anisotropic Pore Characteristics for ECMO Membranes Theoretically Validating the Risk of SARS-CoV-2 Permeation.

The objective of this study is to clarify the pore structure of ECMO membranes by using our approach and theoretically validate the risk of SARS-CoV-2 permeation. There has not been any direct evidence for SARS-CoV-2 leakage through the membrane in ECMO support for critically ill COVID-19 patients. The precise pore structure of recent membranes was elucidated by direct microscopic observation for the first time. The three types of membranes, polypropylene, polypropylene coated with thin silicone layer, and polymethylpentene (PMP), have unique pore structures, and the pore structures on the inner and outer surfaces of the membranes are completely different anisotropic structures. From these data, the partition coefficients and intramembrane diffusion coefficients of SARS-CoV-2 were quantified using the membrane transport model. Therefore, SARS-CoV-2 may permeate the membrane wall with the plasma filtration flow or wet lung. The risk of SARS-CoV-2 permeation is completely different due to each anisotropic pore structure. We theoretically demonstrate that SARS-CoV-2 is highly likely to permeate the membrane transporting from the patient's blood to the gas side, and may diffuse from the gas side outlet port of ECMO leading to the extra-circulatory spread of the SARS-CoV-2 (ECMO infection). Development of a new generation of nanoscale membrane confirmation is proposed for next-generation extracorporeal membrane oxygenator and system with long-term durability is envisaged.

Effects of ruminal administration of soy sauce oil on functional fatty acids in the rumen, blood and milk of dairy cows.

OBJECTIVE: Soy sauce oil, a byproduct of whole soybean processing by the soy sauce industry, was evaluated as a source of linoleic acid for dairy cows for the purpose of manipulating the composition of milk. METHODS: Eight dairy Holstein cows fitted with rumen cannulas were used for ruminal administration of soy sauce oil for a 28-day period using a 4×4 Latin square study design with 4 doses (0, 200, 400, and 600 g soy sauce oil/d). RESULTS: Although dry matter intake and milk yield were not affected by soy sauce oil administration, ruminal concentrations of total volatile fatty acids and acetate were decreased, specifically at 600 g/d administration. While milk fat percentage was decreased with administration of soy sauce oil, proportions of linoleic, vaccenic and conjugated linoleic acids in the rumen, blood and milk were increased with increasing soy sauce oil dose. CONCLUSION: These results suggest that soy sauce oil feeding could be useful for improving milk functionality without adverse effects on animal production performance when fed at less than 400 g/d.

Thermally controlled intracellular uptake system of polymeric micelles possessing poly(N-isopropylacrylamide)-based outer coronas.

Temperature-induced intracellular uptake mechanism of thermoresponsive polymeric micelles comprising poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(d,l-lactide) (P(IPAAm-DMAAm)-b-PLA) inside cultured bovine carotid endothelial cells is investigated by flow cytometry and confocal laser scanning microscopy. Hydrodynamic sizes of P(IPAAm-DMAAm)-b-PLA micelles are approximately 20 nm below the lower critical solution temperature (LCST) of 39.4 degrees C, and their sizes increased to ca. 600 nm above the LCST due to the aggregation of micelles. Intracellular uptake of P(IPAAm-DMAAm)-b-PLA micelles is significantly limited at a temperature below the micellar LCST, 37 degrees C. Of great interest, the P(IPAAm-DMAAm)-b-PLA micelles are internalized into the cells above the micellar LCST (42 degrees C), being dependent on polymer concentration, time, and temperature. By contrast, no intracellular uptake of polyethylene glycol-b-PLA micelles is observed regardless of temperature changes. Enhanced intracellular micelle uptake is probably due to the enhanced interactions between the micelles and cell membranes through the dehydration of corona-forming thermoresponsive polymer chains. Internalization of submicrometer-scale micellar aggregates inside the cells is probably due to their various endocytosis mechanisms. P(IPAAm-DMAAm)-b-PLA micelles localize at the Golgi apparatus and endoplasmic reticulum, but not inside lysosomes. These results indicate that the thermoresponsive polymeric micelles are greatly promising as intracellular delivery tools of drugs, nucleic acids, and peptides/protein without lysosomal decomposition in conjunction with applied heating.

Development of a device for chemiluminescence determination of superoxide generated inside a dialysis hollow-fiber membrane.

Reactive oxygen species (ROS) generated during hemodialysis treatment cause dialysis complications because of the high reactivity of ROS. To prevent dialysis complications caused by oxidative stress, it is important to evaluate the generation and dismutation of ROS during hemodialysis treatment. In this study, our aim was to develop a device to determine superoxide (O(2)(-)) generated inside a dialysis hollow fiber, and also to examine whether this device could detect O(2)(-) separated from plasma using hollow fibers. Experimental apparatus was set up so that hypoxanthine (HX) solution flowed inside the hollow fibers and 2-methyl-6-p-methoxyphenylethynyl-imidazopyrazinone (MPEC) solution flowed outside the hollow fibers. Then, xanthine oxidase (XOD) solution was added to the HX solution to generate O(2)(-), and chemiluminescence resulting from the reaction of O(2)(-) with MPEC was measured with an optical fiber. Chemiluminescence intensity was measured at different HX concentrations, and the peak area of relative luminescence intensity yielded a first-order correlation with the HX concentration. Based on the relationship between HX and O(2)(-) concentrations determined by the cytochrome c reduction method, the relative luminescence intensity measured by this device was linearly dependent on the O(2)(-) concentration inside the hollow fibers. After modifications were made to the device, XOD solution injection into plasma including HX resulted in an increase in the relative luminescence intensity. We concluded that this novel device based on chemiluminescence is capable of determining aqueous O(2)(-) generated inside a hollow fiber and also of detecting O(2)(-) in plasma.

Newly Developed Pediatric Membrane Oxygenator that Suppresses Excessive Pressure Drop in Cardiopulmonary Bypass and Extracorporeal Membrane Oxygenation (ECMO).

This article developes a pediatric membrane oxygenator that is compact, high performance, and highly safe. This novel experimental approach, which imaging the inside of a membrane oxygenator during fluid perfusion using high-power X-ray CT, identifies air and blood retention in the local part of a membrane oxygenator. The cause of excessive pressure drop in a membrane oxygenator, which has been the most serious dysfunction in cardiovascular surgery and extracorporeal membrane oxygenation (ECMO), is the local retention of blood and air inside the oxygenator. Our designed blood flow channel for a membrane oxygenator has a circular channel and minimizes the boundary between laminated parts. The pressure drop in the blood flow channel is reduced, and the maximum gas transfer rates are increased by using this pediatric membrane oxygenator, as compared with the conventional oxygenator. Furthermore, it would be possible to reduce the incidents, which have occurred clinically, due to excessive pressure drop in the blood flow channel of the membrane oxygenator. The membrane oxygenator is said to be the "last stronghold" for patients with COVID-19 receiving ECMO treatment. Accordingly, the specification of our prototype is promising for low weight and pediatric patients.

Validity of Three-Dimensional Tortuous Pore Structure and Fouling of Hemoconcentration Capillary Membrane Using the Tortuous Pore Diffusion Model and Scanning Probe Microscopy.

Hemoconcentration membranes used in cardiopulmonary bypass require a pore structure design with high pure water permeability, which does not allow excessive protein adsorption and useful protein loss. However, studies on hemoconcentration membranes have not been conducted yet. The purpose of this study was to analyze three-dimensional pore structures and protein fouling before and after blood contact with capillary membranes using the tortuous pore diffusion model and a scanning probe microscope system. We examined two commercially available capillary membranes of similar polymer composition that are successfully used in hemoconcentration clinically. Assuming the conditions of actual use in cardiopulmonary bypass, bovine blood was perfused inside the lumens of these membranes. Pure water permeability before and after bovine blood perfusion was measured using dead-end filtration. The scanning probe microscopy system was used for analysis. High-resolution three-dimensional pore structures on the inner surface of the membranes were observed before blood contact. On the other hand, many pore structures after blood contact could not be observed due to protein fouling. The pore diameters calculated by the tortuous pore diffusion model and scanning probe microscopy were mostly similar and could be validated reciprocally. Achievable pure water permeabilities showed no difference, despite protein fouling on the pore inlets (membrane surface). In addition, low values of albumin sieving coefficient are attributable to protein fouling that occurs on the membrane surface. Therefore, it is essential to design the membrane structure that provides the appropriate control of fouling. The characteristics of the hemoconcentration membranes examined in this study are suitable for clinical use.

Temperature-induced intracellular uptake of thermoresponsive polymeric micelles.

Well-defined diblock copolymers comprising thermoresponsive segments of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide) (P(IPAAm-co-DMAAm)) and hydrophobic segments of poly(d,l-lactide) were synthesized by combination of RAFT and ring-opening polymerization methods. Terminal conversion of thermoresponsive segments was achieved through reactions of maleimide or its Oregon Green 488 (OG) derivative with thiol groups exposed by cleavage of polymer terminal dithiobenzoate groups. Thermoresponsive micelles obtained from these polymers were approximately 25 nm when below the lower critical solution temperature (LCST) of 40 degrees C, and their sizes increased to an average of approximately 600 nm above the LCST due to aggregation of the micelles. Interestingly, the OG-labeled thermoresponsive micelles showed thermally regulated internalization to cultured endothelial cells, unlike linear thermoresponsive P(IPAAm-co-DMAAm) chains. While intracellular uptake of P(IPAAm-co-DMAAm) was extremely low at temperatures both below and above the micellar LCST, the thermoresponsive micelles showed time-dependent intracellular uptake above the LCST without exhibiting cytotoxicity. These results indicate that the new thermoresponsive micelle system may be a greatly promising intracellular drug delivery tool.

Computational evaluation of dialysis fluid flow in dialyzers with variously designed jackets.

Dialyzer performance strongly depends on the flow of blood and dialysis fluid as well as membrane performance. It is necessary, particularly to optimize dialysis fluid flow, to develop a highly efficient dialyzer. The objective of the present study is to evaluate by computational analysis the effects of dialyzer jacket baffle structure, taper angle, and taper length on dialysis fluid flow. We modeled 10 dialyzers of varying baffle angles (0, 30, 120, 240, and 360 degrees ) with and without tapers. We also modeled 30 dialyzers of varying taper lengths (0, 12.5, 25.0, and 50.0 mm) and angles (0, 2, 4, and 6 degrees ) based on technical data of APS-SA dialyzers having varying surface areas of 0.8, 1.5, and 2.5 m(2) (Rexeed). Dialysis fluid flow velocity was calculated by the finite element method. The taper part was divided into 10 sections of varying fluid resistances. A pressure of 0 Pa was set at the dialysis fluid outlet, and a dialysis fluid flow rate of 500 mL/min at the dialysis fluid inlet. Water was used as the dialysis fluid in the computational analysis. Results for dialysis fluid flow velocity of the modeled dialyzers indicate that taper design and a fully surrounded baffle are important in making the dialysis fluid flow into a hollow-fiber bundle easily and uniformly. However, dialysis fluid flow channeling occurred particularly at the outflowing part with dialyzers having larger taper lengths and angles. Optimum design of dialysis jacket structure is essential to optimizing dialysis fluid flow and to increasing dialyzer performance.

Development of an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer.

The instability of enzymatic glucose sensors has prevented the development of a practical artificial pancreas for diabetic patients. We therefore developed an enzyme-free glucose sensor using the gate effect of a molecularly imprinted polymer (MIP). This sensor has the advantages of improved stability and a simplified manufacturing procedure. An adduct of glucose and 4-vinylphenylboronic acid (VPBA) was synthesized by esterification and was then purified. The copolymer of the glucose/VPBA adduct and methylene bisacrylamide was grafted onto an indium tin oxide electrode surface. Glucose was washed out from the copolymer to obtain an MIP layer. Cyclic voltammetry of ferrocyanide in aqueous solution was performed using an MIP-grafted electrode, and the effect of glucose on the anodic current intensity was evaluated. The anodic current intensity was sensitive to the glucose concentration, and the dynamic range (0-900 mg/dl) covered the typical range of diabetic blood glucose levels. The response time of the MIP-grafted electrode to a stepwise change in the glucose concentration was approximately 3-5 min. Thus, we can conclude that, by taking advantage of its gate effect, it is feasible to use an MIP-grafted electrode as a glucose sensor for monitoring blood sugar in diabetic patients.

Fluorescence enhancement of fluorescein isothiocyanate-labeled protein a caused by affinity binding with immunoglobulin g in bovine plasma.

Fluorescence enhancement of fluorescein isothiocyanate-labeled protein A (FITC-protein A) caused by the binding with immunoglobulin G (IgG) in bovine plasma was studied. FITC-protein A was immobilized onto a glass surface by covalent bonds. An increase in fluorescence intensity was dependent on IgG concentration ranging from 20 to 78 µg/mL in both phosphate buffer saline and bovine plasma. This method requires no separation procedure, and the reaction time is less than 15 min. A fluorescence enhancement assay by the affinity binding of fluorescence-labeled reagent is thus available for the rapid determination of biomolecules in plasma.

The effect of extensible PEG tethers on shielding between grafted thermo-responsive polymer chains and integrin-RGD binding.

The affinity control of integrin-RGD (Arg-Gly-Asp) binding by a thermal "on-off" switch has been achieved using newly designed surfaces presenting grafted temperature-responsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) copolymers functionalized with synthetic peptides. The prepared surface was designed to expose the tethered peptides available for cell binding at active "on" state above the lower critical solution temperature (LCST). The fully extended chains, on the other hand, masked the peptides completely and the cells started to detach from the surfaces at inactive "off" sate below the LCST. This paper elucidates the shielding effect of the grafted polymer chains on the dissociation of integrin-RGD binding below the LCST. To assess the ability of the polymer-shielding, extensible poly(ethylene glycol) (PEG) tethers were introduced between peptides and the grafted polymers. PEG chains allow peptides to be tethered to surfaces via functional PEG end-groups, leading to active "on" state even below the LCST. The time required to release cells from the surface was found to be longer when peptides were coupled to an extensible tether ends, suggesting that the surfaces can engender cell attachment through adhesive moieties covalently bound to the free ends of PEG chains. These results indicate that architectural changes on the nanometer length scale are crucial for controlling integrin-RGD binding and one of the main factors causing cell detachment is the shielding effect of the grafted polymer chains.