"Smart" mobile affinity matrix for microfluidic immunoassays

There is a current need for simple methods for immobilizing biomolecules within microfluidic channels. Here, a technique is reported for reversibly immobilizing immunoassay components in a channel zone that can be simply controlled by integrated heating elements. Latex beads were modified with the temperature-responsive polymer poly(N-isopropylacrylamide)(PNIPAAm) and co-modified with biotinylated poly(ethylene glycol)(PEG). PNIPAAm undergoes a hydrophilic-to-hydrophobic transition when the temperature is raised above the lower critical solution temperature (LCST)( approximately 28 degrees C in the solutions used here). This reversible transition drives the aggregation and dis-aggregation of the modified beads in heated zones within poly(ethylene terephthalate)(PET) microchannels. Biotinylated monoclonal antibodies for the drug digoxin were bound via streptavidin to the biotin-PEG-coated beads. These antibody-functionalized beads were then reversibly immobilized by aggregation and hydrophobic adhesion to the surface of PET microfluidic channels in response to a thermal stimulus. The antibodies on the beads immobilized in the channel were shown to bind digoxin and a competitor fluorescent ligand from a flow stream in a quantitative competitive assay format that reported the digoxin concentration. The antibodies could be replenished for each immunoassay trial, using the reversible, temperature-controlled immobilization process. This technique allows reagent immobilization immediately prior to an analytical procedure, following the removal of previously utilized beads, guaranteeing fresh and active immobilized biomolecules. Furthermore, it provides a simple approach to multiplexing through the simultaneous or sequential injection of different antibody-coated bead species, potentially at multiple sites in the integrated device channels.     

Thermoreversible gelation on cooling and on heating of an aqueous gelatin–poly(N-isopropylacrylamide) conjugate

A polymer conjugate composed of 43 wt% gelatin and 57 wt% poly(N-isopropylacrylamide) (PNIPAAm) was prepared. The dynamic viscoelastic properties of an aqueous solution of the conjugate at the concentration of 5 wt% were examined. The solution was viscous fluid at 30°C and turned into an elastic homogeneous hydrogel upon heating above 34°C or upon cooling below 10°C. The resultant hydrogels turned back into a solution at the opposite temperature cycles of the gelation. It is considered that the driving force of the gelation is the intermolecular hydrophobic interaction of PNIPAAm blocks or the intermolecular helix association of gelatin blocks, respectively, on heating or on cooling. © 1998 John Wiley & Sons, Ltd.     

Study of temperature-responsibility on the surfaces of a thermo-responsive polymer modified stationary phase

We investigated a thermo-sensitive polymer, poly(N-isopropylacrylamide) (PNIPAAm), which is the basis of an HPLC stationary phase. We prepared a PNIPAAm terminally-modified surface. In this study, we investigated the effect of PNIPAAm on the surface of a stationary phase on separation based on changes of the retention time with the temperature step gradient. As the temperature changed the surface property of the stationary phase switched from hydrophilic to hydrophobic. The retention on the polymer-modified stationary phase remarkably changed upon changing the temperature. Using a column packed with PNIPAAm-modified silica, the separation of steroids was carried out by changing the temperature. With increasing temperature, an increased interaction between solutes and PNIPAAm-grafted surfaces of the stationary phases was observed. A temperature-dependent resolution of steroids was achieved using only water as a mobile phase. The PNIPAAm-modified surface of the stationary phase exhibited temperature-controlled hydrophilic-hydrophobic changes. The drastic and reversible surface hydrophilic-hydrophobic property alteration for PNIPAAm terminally-grafted surfaces should be due to rapid changes in the polymer hydration state around the polymer's transition temperature. A solvent gradient elution-like effect could be achieved with a single mobile phase by programmed temperature changes during chromatographic runs. This system should be highly useful to control the function and property of the stationary phase for HPLC only by changing the temperature with an aqueous solvent.     

Temperature-responsive stationary phase utilizing a polymer of proline derivative for hydrophobic interaction chromatography using an aqueous mobile phase

A new method of chromatography is proposed, utilizing a thermo-responsive polymer carrying an amino acid ester residue for the stationary phase of high-performance liquid chromatography (HPLC). We have been investigating the new concept of chromatography, a temperature-responsive chromatography, using temperature-responsive poly(N-isopropylacrylamide) (PNIPAAm)-modified surface for HPLC with a constant aqueous media as the mobile phase. In this study, we designed and synthesized thermo-responsive poly(acryloyl-L-proline methyl ester) and its copolymer with N-isopropylacrylamide (NIPAAm). Homopolymers of acryloyl-L-proline methyl ester and copolymer were prepared by the reaction of radical telomerization. These polymers underwent a reversible phase transition from water-soluble forms into aggregates by changing the temperature, similar to PNIPAAm. The surface properties and functions of stationary phases modified with poly(acryloyl-L-proline methyl ester) were controlled by the external temperature. In the chromatographic system, we separated steroids and amino acids with a variety of hydrophobicities using a sole aqueous mobile phase. In contrast to a PNIPAAm-modified surface, a poly(acryloyl-L-proline methyl ester)-modified surface showed a greater affinity for hydrophobic amino acids.     

Temperature-responsive chromatography for the separation of biomolecules

Temperature-responsive chromatography for the separation of biomolecules utilizing poly(N-isopropylacrylamide) (PNIPAAm) and its copolymer-modified stationary phase is performed with an aqueous mobile phase without using organic solvent. The surface properties and function of the stationary phase are controlled by external temperature changes without changing the mobile-phase composition. This analytical system is based on nonspecific adsorption by the reversible transition of a hydrophilic-hydrophobic PNIPAAm-grafted surface. The driving force for retention is hydrophobic interaction between the solute molecules and the hydrophobized polymer chains on the stationary phase surface. The separation of the biomolecules, such as nucleotides and proteins was achieved by a dual temperature- and pH-responsive chromatography system. The electrostatic and hydrophobic interactions could be modulated simultaneously with the temperature in an aqueous mobile phase, thus the separation system would have potential applications in the separation of biomolecules. Additionally, chromatographic matrices prepared by a surface-initiated atom transfer radical polymerization (ATRP) exhibit a strong interaction with analytes, because the polymerization procedure forms a densely packed polymer, called a polymer brush, on the surfaces. The copolymer brush grafted surfaces prepared by ATRP was an effective tool for separating basic biomolecules by modulating the electrostatic and hydrophobic interactions. Applications of thermally responsive columns for the separations of biomolecules are reviewed here.     

Protecting enzymes against heat inactivation by temperature-sensitive polymer in confined space

At high temperature, many enzymes are inactivated by aggregations at hydrophobic sites which are exposed on denaturation. Isolating denatured enzymes via hydrophobic interactions with other material is a significant method to prevent enzymes from aggregation. But the temperature-sensitive polymer poly(N-isopropylacrylamide) (PNIPAAm), supposed to protect enzymes spontaneously at high temperatures, can not efficiently complex denatured carbonic anhydrase B (CAB, as a model enzyme) in bulk aqueous solution due to different phase transition speeds. Here, we present a novel method for protecting enzymes against heat inactivation, in which PNIPAAm and CAB are encapsulated in a confined space constructed by reverse microemulsion. At high temperatures, PNIPAAm forms nanoscale aggregates possessing both large specific surface areas and hydrophobic surfaces, and then adsorbs denatured CAB via hydrophobic interactions to avoid intermolecular aggregation of CAB. With cooling, CAB is released spontaneously and recovers its activity. The assays for enzymatic activity demonstrate that CAB is effectively protected against heat inactivation through this method (protection efficiency is up to 83.2%).     

Polymer-mediated extraction of 8-quinolinolato metal chelates for the determination of metal ions in water with graphite furnace atomic absorption spectrometry

Water-insoluble 8-quinolinolato metal chelates were formed and were stably solubilized in the aqueous solution of a water-soluble polymer, poly (N-isopropylacrylamide)(PNIPAAm), at room temperature. When the solution was heated at 50 degrees C, PNIPAAm precipitated and then formed a gum-like aggregate (polymer phase) having a very small volume. Accompanying the polymer precipitation, hydrophobic 8-quinolinolato chelates with cobalt(II), iron(III), nickel(II), and copper(II) ions were efficiently incorporated into the polymer phase. At 0.5% (w/v) of PNIPAAm and 8.0 mM of 8-quinolinol, the recoveries in the incorporation of four metal chelates were quantitative. The fluorescence spectra of a probe suggests that the hydrated polymer in the aqueous solution provides hydrophobic portions which can incorporate hydrophobic metal chelates. The polymer phase was easily taken out from the solution and was dissolved with a small amount of acetonitrile. The resulting solution could be directly introduced into a graphite furnace of atomic absorption spectrometry. The signal intensities for the absorbance of cobalt after concentrating the chelate were 100-fold greater than those before the concentration.     

P(NIPAM-co-NVP)共聚物分子链水溶液中的可逆聚集研究

用溶液聚合方法合成了线型聚(N-异丙基丙烯酰胺-co-N-乙烯基吡咯烷酮)共聚物,通过弹性光散射(elastic light scattering,ELS)、荧光光谱与动态光散射研究了共聚物水溶液分子链可逆聚集的温度和时间依赖性.研究表明,升温时,ELS强度增加,分子链聚集;降温时,ELS强度降低,聚集的分子链解离.荧...     

Heat-induced phase transitions from an aqueous solution to precipitates in a poly(sodium 4-styrenesulfonate)/tetradecyltrimethylammonium bromide system

For the first time, temperature-induced phase transitions upon heating and cooling an aqueous solution that contained oppositely charged polyelectrolyte and surfactant mixtures was observed. The phase transition from micelles to vesicles, then to the coexistence of vesicles and superstructures that have the morphology of melon seeds, and finally to precipitates was determined by means of turbidity measurements and transmission electron microscopy images. These phase transitions were shown to be reversible and reproducible after several heating and cooling cycles were performed on the same sample. The novel observations for the temperature-induced phase transition from primary aggregates, such as micelles, to superstructures (i.e., vesicles) should provide new understanding for surfactant sciences, and in particular for self-assembled amphiphilic systems.     

Thermoresponsive micelles from well‐defined block copolymers synthesized via reversible addition–fragmentation chain transfer polymerization

Poly(N‐isopropylacrylamide) (PNIPAAm) homopolymers synthesized by reversible addition–fragmentation chain transfer polymerization were used as macro‐chain‐transfer agents to synthesize smart amphiphilic block copolymers with a switchable hydrophilic–hydrophobic block of PNIPAAm and a hydrophilic block of poly(N‐dimethylacrylamide). All polymers were characterized by gel permeation chromatography, ¹H NMR, and differential scanning calorimetry. The reversible micelles formed by the block copolymers of various compositions in aqueous solutions were characterized by ¹H NMR, dynamic light scattering, and tensiometry. Micelles were observed in the aqueous solutions when the temperature was increased to 40 °C because of the collapse of the PNIPAAm structure, which led to a PNIPAAm hydrophobic block. The drug loading capacity was illustrated with the use of the solvatochromic Reichardt's dye and measured by ultraviolet–visible. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3643–3654, 2005     

Anion effects on the phase transition of N‐isopropylacrylamide hydrogels

NMR spectra were collected for poly(N‐isopropylacrylamide) (PNIPAAm) hydrogel using high‐resolution magic angle spinning (HRMAS) after gel pieces were hydrated in the presence of D₂O, NaF, NaCl, and NaI aqueous solutions. Changes in the peak height intensity of the spectra provide quantitative insight into the phase transition process. The thermodynamic values of the phase transition were calculated using a van't Hoff analysis of the NMR data. Unlike the trend observed for decreases in the (LCST), changes in the enthalpy and entropy did not clearly display a linear dependence with respect to salt concentration. Rather, it was observed that increases in salt concentration did not affect the enthalpy and entropy to the extent as the initial change observed between no salt and 100 mM solutions. Finally, the effect of salts on the hysteresis of the rehydrating process was observed. Hysteresis occurs due to the need for hydrophobic interactions to break down before water is able to infiltrate the polymer matrix. NaF stabilizes hydrophobic interactions while NaI destabilize hydrophobic interactions, causing them to break down at higher temperatures. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

FtsZ Reorganization Facilitates Deformation of Giant Vesicles in Microfluidic Traps**

The geometry of reaction compartments can affect the local outcome of interface-restricted reactions. Giant unilamellar vesicles (GUVs) are commonly used to generate cell-sized, membrane-bound reaction compartments, which are, however, always spherical. Herein, we report the development of a microfluidic chip to trap and reversibly deform GUVs into cigar-like shapes. When trapping and elongating GUVs that contain the primary protein of the bacterial Z ring, FtsZ, we find that membrane-bound FtsZ filaments align preferentially with the short GUV axis. When GUVs are released from this confinement and membrane tension is relaxed, FtsZ reorganizes reversibly from filaments into dynamic rings that stabilize membrane protrusions; a process that allows reversible GUV deformation. We conclude that microfluidic traps are useful for manipulating both geometry and tension of GUVs, and for investigating how both affect the outcome of spatially-sensitive reactions inside them, such as that of protein self-organization.     

Thermosensitivity of optically active hydrogels constructed with N-(L)-(1-hydroxymethyl)propylmethacrylamide

We previously reported on the water soluble, optically active polymer, poly[N-(L)-(1-hydroxymethyl)propylmethacrylamide] (P(L-HMPMA)), which has lower critical solution temperatures of approximately 30 and 21 degrees C upon heating and cooling, respectively. The phase separation behavior of the P(L-HMPMA)-water binary system has a reversible and clear hysteresis during heating and cooling cycles. The present study describes the thermosensitive properties of the optically active polymer and its hydrogel. Circular dichroism and microcalorimetric measurements of the polymer in water supported the hypothesis that the soluble polymer chains might be compactly folded with an interaction between optically active side chains. In addition, these measurements showed the polymer chains in a state of relatively low hydration compared to that of P(D,L-HMPMA), which was free-radically synthesized from racemates of monomers. The solution properties reflected the swollen-shrunken behavior of corresponding hydrogels in response to temperature changes. The microscopic observation of aqueous polymer solutions and hydrogels also confirmed that the optically active properties of polymer chains affect their structure and thermosensitivity. SEM micrograph of the surfaces of the crosslinked P(L-HMPMA).     

Thermosensitive graphene nanocomposites formed using pyrene‐terminal polymers made by RAFT polymerization

Thermosensitive graphene‐polymer composites have been prepared by attaching poly(N‐isopropylacrylamide) (PNIPAAm) onto the basal plane of graphene sheets via π‐π stacking. Pyrene‐terminated PNIPAAm was synthesized using reversible addition fragmentation chain transfer (RAFT) polymerization via a pyrene‐functional RAFT agent. Aqueous solutions of the graphene‐polymer composites were stable and thermosensitive. The lower critical solution temperature (LCST) of pyrene‐terminated PNIPAAm was measured to be 33 °C. When the pyrene‐functional polymer was attached to graphene the resultant composites were also thermosensitive in aqueous solutions exhibiting a reversible suspension behavior at 24 °C. Atomic force microscopy (AFM) analysis revealed that the thickness of a graphene‐PNIPAAm (M_n: 10,000 and PDI: 1.1) sheet was ～5.0 nm. The surface coverage of polymer chains on the graphene basal plane was calculated to be 7.2 × 10^?11 mol cm^?2. The graphene‐PNIPAAm composite material was successfully characterized using X‐ray photoelectron spectroscopy (XPS), attenuated total reflection infrared (ATR‐IR) spectroscopy, and thermogravimetric analysis (TGA). © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 425–433, 2010     

Unusual phase behavior in mixtures of poly(ethylene oxide) and ethyl alcohol

Poly(ethylene oxide) (PEO), soluble in both aqueous and organic solvents, is one of the most intriguing polymers. PEO solution properties have been extensively studied for decades; however, many of the studies have focused on specific properties, such as clustering, of PEO in aqueous solutions, and the behavior of PEO in organic solvents has not been adequately explored. The results presented here demonstrate that PEO crystallizes into a lamellar structure in ethyl alcohol after the mixture is quenched to room temperature from a temperature above the crystal melting point. Above the melting temperature, PEO completely dissolves in ethyl alcohol, and the mixture exhibits regular polymer solution thermodynamic behavior with an upper critical solution temperature (UCST) phase diagram. Remarkably, the UCST phase boundary is significantly below the melting temperature, and this indicates that the system undergoes a crystallization process before the phase separation can occur upon cooling and, therefore, possesses an unusual phase transition. The phase transition from the crystalline state to the miscible solution state is reversible upon heating or cooling and can be induced by the addition of a small amount of water. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 557–564, 2006     

Vesicle fission of giant unilamellar vesicles of liquid-ordered-phase membranes induced by amphiphiles with a single long hydrocarbon chain

Vesicle fissions are very important processes of biomembranes in cells, but their mechanisms are not clear and are controversial. Using the single giant unilamellar vesicle (GUV) method, we recently found that low concentrations (less than the critical micelle concentration (CMC)) of lysophosphatidylcholine (lyso-PC) induced the vesicle fission of GUVs of dipalmitoylphosphatidylcholine/cholesterol(6/4) (DPPC/chol(6/4)) membranes and sphingomyelin/cholesterol membranes (6/4) in the liquid-ordered (lo) phase. In this report, to elucidate its mechanism, we have investigated the effect of low concentrations (much less than their CMC) of other amphiphiles with a single long hydrocarbon chain (i.e., single long chain amphiphiles) on DPPC/chol(6/4) GUVs as well as the effect of the membrane composition on the lyso-PC-induced vesicle fission. We found that low concentrations of single long chain amphiphiles (lyosophosphatidic acid, octylglucoside, and sodium dodecyl sulfate) induced the shape change from a prolate to two spheres connected by a very narrow neck, indicating that the single long chain amphiphiles can be partitioned into the external monolayer in the lo phase of the GUV from the aqueous solution. As the single long chain amphiphile concentrations were increased, all of them induced vesicle fission of DPPC/chol(6/4) GUVs above their threshold concentrations. To elucidate the role of cholesterol in the single long chain amphiphile-induced vesicle fission, we investigated the effect of lyso-PC on GUVs of dioleoyl-PC (DOPC)/chol(6/4) membranes in the Lalpha phase; no vesicle fission occurred, indicating that cholesterol in itself did not play an important role in the vesicle fission. Finally, to elucidate the effect of the inclusion of DOPC in the lo-phase membrane of GUVs on the lyso-PC-induced vesicle fission of the DPPC/chol(6/4) GUV, we investigated the effect of low concentrations of lyso-PC on GUVs of DPPC/DOPC/chol membranes. With an increase in DOPC concentration in the membrane, the threshold concentration of lyso-PC increased. At and above 30 mol % DOPC, no vesicle fission occurred. On the basis of these results, we have proposed a hypothesis of the mechanism of the single long chain amphiphile-induced vesicle fission of a GUV of a lo-phase membrane.     

Phase diagrams of a hydroxypropyl cellulose-water system under static conditions and in the shear field

Phase diagrams have been constructed for a hydroxypropyl cellulose-water system under static conditions and in a shear field by the cloud-point method and a transmitted polarized light intensity measurements. As the molecular mass of the polymer is increased, the curve delimiting isotropic and anisotropic solutions shifts to lower concentrations. Deformation causes changes in the phase transition temperatures of solutions both upon heating and cooling.     

Synthesis and characterization of reversible thermosensitive methylated chitin and its application in zinc-ion battery thermal runaway

Novel water-soluble methylated chitins (MCHs) were synthesized homogeneously in aqueous alkaline solution. The relatively mild reaction conditions resulted in the MCH with high degree of acetylation (DA >0.76). The chemical structure of the obtained MCHs was analyzed and the degree of methylation substitution (DS) and DA were determined by proton NMR in both D₂O and 20% DCl/D₂O. The MCH aqueous solutions (DS = 0.46 ~ 0.71) showed a reversible thermosensitive sol–gel–sol transition upon heating and cooling. The gel transition temperature of these MCHs (in the range of 15–85 °C) increased with increasing DS and decreasing polymer concentration. Thermal runaway has been an important safety issue impeding the development of high-energy-density zinc-ion batteries. A smart thermosensitive reversible electrolyte was prepared based on this MCH for the aqueous zinc-ion battery to prevent thermal runaway. When the temperature of zinc-ion battery rises or even gets out of control, the thermosensitive electrolyte can quickly gel and inhibit the migration of zinc ions, resulting in increase of the internal resistance and realizing intelligent and efficient thermal self-protection. Thus the novel thermosensitive methylated chitin shows promise for safe aqueous zinc-ion batteries.     

Thermoresponsive transport through ordered mesoporous silica/PNIPAAm copolymer membranes and microspheres

Thermosensitive inorganic-organic hybrid polymers and gels can be used for controlled molecular transport in a variety of applications that require robust, mechanically stable materials. Silica and poly(N-isopropylacrylamide) (PNIPAAm) precursors were copolymerized in the presence of surfactant supramolecular assemblies to form hybrid gels with ordered nanostructure. This method was less complicated and results in enhanced reversible transport properties compared to previous approaches noted herein. In this study, the thermoresponsive polymer, PNIPAAm, was incorporated into polymerizing silica networks using the coupling agent 3-methacryloxypropyltrimethoxysilane. The hydration transition of PNIPAAm associated with its lower critical solution temperature (LCST) in aqueous solution was retained in the hydrated silica matrices and was used to control the permeability of membranes and molecular release behavior of particles. This report presents new methods for formation of hybrid silica/PNIPAAm membranes and particles, characterization of these materials, and documentation of reversible molecular transport properties of these new hybrid materials.     

Thermotropic liquid crystals embedded in a high water gel system

An emulsion was formed when the thermotropic liquid crystal (LC) mixture E5 was added to an aqueous polyvinylalcohol (pva) solution and shaken. This emulsion was gelled by addition of an aqueous borax solution. The pva polymer functioned not only as the gelling agent but also appeared to act as a polymeric surfactant which stabilised the LC droplets. This high water gel-liquid crystal (HWG-LC) system contained nearly 80 wt % water and more LC wt % than polymer. The system was thermally reversible, undergoing a gel to sol transition upon heating to 70°C and reforming a gel upon cooling. The HWG-LC showed electrooptical behaviour dependent upon a switched electric field when constrained between transparent electrodes. The pressure required to form a thin film between these electrodes induced a structural emulsion in the dispersion causing LC droplet disruption and the formation of an LC network in the gel.