3-acrylamidophenylboronic Acid Research Articles

Boronate-Containing Copolymer Grafted on Eupergit C as Matrix for Affinity Chromatography: Isotherms and Kinetics Study

Eupergit C, an acrylic polymer support containing oxirane groups, for pseudo affinity adsorption of bioproducts was modified and functionalized by 1,2-ethanedithiol followed by graft copolymerization of 3-acrylamidophenylboronic acid and N,N-dimethylacrylamide. The resin was characterized by elemental analysis and IR spectroscopy and was subjected to biochemical evaluation through batch binding and column chromatography of mucin. Based on equilibrium adsorption data the Langmuir and Freundlich constants were determined 0.04 and 0.84 at pH 8 and 0.19 and 4.53 at pH 8.5, respectively. Subsequently adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetics equation. It was shown that the pseudo-second-order kinetic equation could best describe adsorption. The generic application of such copolymer grafted for the adsorption and separation of bioproducts is discussed.

Measurement of Glucose in Blood with a Phenylboronic Acid Optical Sensor

Current methods of glucose monitoring rely predominantly on enzymes such as glucose oxidase for detection. Phenylboronic acid receptors have been proposed as alternative glucose binders. A unique property of these molecules is their ability to bind glucose in a fully reversible covalent manner that facilitates direct continuous measurements. We examined (1) the ability of a phenylboronic-based sensor to measure glucose in blood and blood plasma and (2) the effect on measurement accuracy of a range of potential interferents. We also showed that the sensor is able to track glucose fluctuations occurring at rates mimicking those experienced in vivo. In vitro static measurements of glucose in blood and blood plasma were conducted using holographic sensors containing acrylamide, N,N'-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl) trimethylammonium chloride. The same sensors were also used for in vitro measurements performed under flow conditions. The opacity of the liquid had no affect on the ability of the optical sensor to measure glucose in blood or blood plasma. The presence of common antibiotics, diabetic drugs, pain killers, and endogenous substances did not affect the measurement accuracy, as shown by error grid analysis. Ex vivo flow experiments showed that the sensor is able to track changes accurately in concentration occurring in real time without lag or evidence of hysteresis. The ability of phenylboronic acid sensors to measure glucose in whole blood was demonstrated for the first time. Holographic sensors are ideally suited to continuous blood glucose measurements, being physically and chemically robust and potentially calibration free.

Continuous Blood Glucose Monitoring with a Thin-Film Optical Sensor

We recently described a holographic optical sensor with improved selectivity for glucose over fructose that was based on a thin-film polymer hydrogel containing phenylboronic acid receptors. The aim of the present work was to measure glucose in human blood plasma as opposed to simple buffers and track changes in concentration at a rate mimicking glucose changes in vivo. We used holographic sensors containing acrylamide, N,N'-methylenebisacrylamide, 3-acrylamidophenylboronic acid, and (3-acrylamidopropyl)trimethylammonium chloride to measure 7 human blood plasma samples at different glucose concentrations (3-33 mmol/L) in static mode. Separately, using a flow cell, the glucose concentration was varied at approximately 0.17-0.28 mmol(-1) x L(-1) x min(-1), and the sensor's ability to continuously monitor glucose was investigated over an extended period. We subjected the results of the ex vivo static measurements to error grid analysis. Of 46 measurements, 42 (91.3%) fell in zone A of a Clarke error grid, and the remainder (8.7%) fell in zone B. The ex vivo flow experiments showed that the sensor is able to accurately track changes in concentration occurring in real time without lag or evidence of hysteresis. We demonstrate the ability of a phenylboronic acid-based sensor to measure glucose in human blood plasma for the 1st time in vitro. Holographic glucose sensors can be used without recourse to recalibration. Their robust nature, coupled with their format flexibility, makes them an attractive alternative to conventional electrochemical enzyme-based methods of glucose monitoring for people with diabetes.

Recognition of sugars on surface-bound cap-shaped gold particles modified with a polymer brush

A dithiolated random copolymer with pendent phenylboronic acid residues (Cys-Poly(3-acrylamidophenylboronic acid- co-2-dimethylaminopropyl methacrylamide), Cys-Poly(APBA- co-DMAPMA)) obtained by photo-iniferter method was accumulated as a polymer brush on a cap-shaped gold particles deposited on a vacuum-evaporated gold film, and the usefulness of the polymer brush as a sensing element for glycoprotein, ovalbumin (OVA), was examined by using UV–vis spectroscopy with a help of surface plasmon resonance. A similar system was constructed with a dithiolated mannose-carrying polymer, dithiolated-poly(2-methacryloyloxyethyl- D-mannopyranoside) (DT-PMEMan), prepared by the atom transfer radical polymerization (ATRP). The brush composed of this polymer was examined as a sensing element for lectin, concanavalin A (Con A). The sensor cells modified with Cys-Poly(APBA- co-DMAPMA) and DT-PMEMan showed a concentration-dependent binding of OVA and Con A, respectively, with a comparable detection limit to those with a monolayer of polymer brush-coated gold particle deposited on a glass substrate. Using this system, it can be expected to open a new perspective to various functional polymer brushes fixed to the cap-shaped gold particle on a solid substrate.

Activation of lymphocyte proliferation by boronate-containing polymer immobilised on substrate: The effect of boron content on lymphocyte proliferation

This study demonstrates that boronic acid-containing polymers coated onto solid support function as synthetic mitogens for mouse lymphocytes. The polymer was synthesized by radical copolymerization of 3-acrylamidophenylboronic acid with dimethylacrylamide (poly(AAPBA-DMAA)). The boronic acid in the trigonal form in the copolymer activated lymphocytes, probably by crosslinkage to glycoprotein moieties on the plasma membrane surface, as in the case of lectin stimulation. A higher concentration of phenylboronic acid on the copolymer surface resulted in greater activation of lymphocytes, suggesting that the number of phenylboronic acid residues per unit area may be a crucial factor in lymphocyte proliferation. The proliferative response of lymphocytes was also affected by the surface wettability, probably due to a difference in the flexibility of polymer strands at the cell-polymer interface.

Holographic Lactate Sensor

Measurement of blood l-lactate is used to assess and monitor exercise performance in sports medicine. This report describes the initial development of a holographic sensor, which employs a synthetic receptor, to enable the selective and continuous real-time measurement of l-lactate for eventual in vivo application. Three boronic acid-based receptors have been synthesized, integrated into thin acrylamide hydrogel films, and then subsequently transformed into holographic sensors. Changes in the replay wavelength of the sensors were used to characterize the swelling behavior of the matrix as a function of l-lactate concentration. It was found that the incorporation of 3-acrylamidophenyl boronic acid into an acrylamide hydrogel produced the largest response toward l-lactate. The effects of hydrogel composition, fluctuating l-lactate concentrations, and the response of potential interfering agents to the sensor have been investigated.

Glucose-sensitive holographic sensors for monitoring bacterial growth.

A glucose sensor comprising a reflection hologram incorporated into a thin, acrylamide hydrogel film bearing the cis-diol binding ligand, 3-acrylamidophenylboronic acid (3-APB), is described. The diffraction wavelength (color) of the hologram changes as the polymer swells upon binding cis-diols. The effect of various concentrations of glucose, a variety of mono- and disaccharides, and the alpha-hydroxy acid, lactate, on the holographic response was investigated. The sensor displayed reversible changes in diffraction wavelength as a function of cis-diol concentration, with the sensitivity of the system being dependent on the cis-diol tested. The effect of varying 3-APB concentration in the hydrogel on the holographic response to glucose was investigated, and maximum sensitivity was observed at a functional monomer concentration of 20 mol %. The potential for using this holographic sensor to detect real-time changes in bacterial cell metabolism was demonstrated by monitoring the germination and subsequent vegetative growth of Bacillus subtilis spores.

Holographic glucose sensors

A novel holographic sensor system capable of detecting dynamic changes in glucose concentration has been developed. The hologram is recorded within a bio-compatible hydrogel matrix containing phenylboronic acid derivatives. On binding glucose, the colour of the hologram red-shifts to longer wavelengths as the hydrogel expands and this colour change is used to quantify glucose concentration. However, phenylboronic acids are non-selective and bind a wide variety of cis-diols. In blood, glucose is the only sugar found free at high concentration, whilst other sugars are typically found as part of glycoproteins and macromolecular structures. Although glycoproteins have been shown to have no effect on the sensor, phenylboronic acids can bind lactate much more readily than glucose. We have designed two polymer hydrogel systems to increase the selectivity of the sensor for glucose over lactate. The first involved the use of high concentrations of 3-acrylamidophenylboronic acid (3-APB) whilst the second system utilised 2-acrylamido-5-fluorophenylboronic acid (5-F-2-MAPB). Both systems displayed an increased selectivity to glucose over lactate at physiological pH and ionic strength and could be deployed as selective holographic sensors for glucose detection in physiological fluids.

Accumulation of phenyl boronic acid-carrying telomers on a gold surface

By coupling two equivalents of N, N-diethyldithiocarbamoylmethylbenzoic acid succinimidyl ester with cystamine dihydrochloride, a disulfide-carrying compound (Cys-BDC) was prepared and used as iniferter (a compound which pursues initiation, chain trans fer, and termination) in the telomerization of N, N-dimethylacrylamide and 3-acrylamidophenylboronic acid. The telomerization was carried out in the presence of N, N, N′, N′-tetraethylthiuram disulfide under photoirradiation at 365 nm. An aqueous solution of the telomer obtained showed responsiveness to both temperature and sugars as confirmed by the turbidity measurements. The telomer formed a self-assembled monolayer (SAM) on a gold surface as confirmed by both cyclic voltammetry (CV) and ellipsometry. The recognition of sugar residues by the telomer SAM constructed on the gold electrode was detected by the CV measurements. The usefulness of the iniferter to prepare various telomer-carrying SAMs with bio-related functions was strongly suggested.

Glucose-Sensitive Inverse Opal Hydrogels: Analysis of Optical Diffraction Response

A glucose-sensitive inverse opal hydrogel was synthesized through photopolymerization of 2-hydroxyethyl methacrylate and 3-acrylamidophenylboronic acid within the interstitial space of a dried poly(styrene) colloidal crystal template, followed by template removal. Charged complex formation between the phenylboronic acid functional group and the 1,2-cis-diol glucose resulted in reversible swelling of the inverse opal hydrogel, which was observed through shifts in the optical diffraction wavelength. The hydrogel was sensitive to glucose at physiological concentrations and ionic strength. The effects of phenylboronic acid concentration, ionic strength, and buffer pH on the equilibrium hydrogel swelling were also studied. The kinetics of hydrogel swelling was also examined, and it was found that the rate of diffraction shift matched well with diffusion-limited kinetics. Additionally, the diffraction response was compared with simulations using the scalar wave approximation and transfer matrix method.

Swelling and Shrinking Kinetics of Totally Synthetic, Glucose-Responsive Polymer Gel Bearing Phenylborate Derivative as a Glucose-Sensing Moiety

A glucose-responsive polymer gel bead with a diameter in the range of several hundred micrometers, bearing a phenylborate derivative (3-acrylamidophenylboronic acid) as a glucose-sensing moiety, was successfully prepared by inverse phase suspension polymerization. The kinetics of the glucose responsive swelling and shrinking process of the gel bead was studied by monitoring changes in the size and shape of the gel under a microscope. The equilibrium swelling volumes of the gel determined under various temperatures and glucose concentrations revealed the presence of critical temperatures and glucose concentrations to induce discontinuous volume phase transitions of the gel. The glucose-induced swelling process was accompanied by the appearance of a marked swelling boundary (swelling front) intervening between the core collapsed phase and the outer swollen layer of the gel, and the swelling curve as a function of the square root of the time exhibited a sigmoidal (nonlinear) feature, both indicating that the...

Characterization of the Swelling of Acrylamidophenylboronic Acid−Acrylamide Hydrogels upon Interaction with Glucose by Faradaic Impedance Spectroscopy, Chronopotentiometry, Quartz-Crystal Microbalance (QCM), and Surface Plasmon Resonance (SPR) Experiments

A m-acrylamidophenylboronic acid−acrylamide copolymer is assembled by electropolymerization on Au-surfaces (Au-electrodes, Au-quartz crystals or Au-glass slides). The electrolysis time controls the film thickness on the electrodes. Addition of glucose to the copolymer film leads to the ligation of the sugar to the boronic acid sites and results in the swelling of the polymer. Faradaic impedance spectroscopy, chronopotentiometry, surface plasmon resonance spectroscopy (SPR), and microgravimetric quartz-crystal-microbalance measurements (QCM) are employed to characterize the swelling of the polymer film upon the binding of glucose. The swelling rate constant, upon the association of glucose to the polymer film, is estimated to be ksw = 1.7 × 10-4 s-1, while the shrinking rate constant of the polymer film, upon depletion of glucose, is ksh = 2.3 × 10-5 s-1. By following the swelling degree of the polymer film at variable glucose concentrations, the polymer matrix is used as an active medium for the sensing o...

Nucleotide-responsive gel tips for atomic force microscopy

A cantilever used for atomic force microscopy (AFM) is modified by an ultra-thin layer of polymeric gel so that the resulting tip can respond differently depending on the kind of nucleotides present in solution. A cross-linked poly((3-acrylamidopropyl)trimethylammonium-co-(3-acrylamido)phenylboronic acid) is grafted on the surface of ordinary AFM tips by in-situ polymerization. The thickness of a dried polymer layer is determined to be 8 nm. In pure water, the force–distance curves of the gel tip against a hydrophobic surface indicate that the gel layer is swelled. At pH 7 and higher, adenosine 5’-monophosphate (AMP) causes the gel to shrink and a strong adhesion to appear in the force curve whereas 2’-deoxyadenosine 5'monophosphate (dAMP) hardly affects the gel layer other than a slight shrinkage. Lowering pH to 4.8 using acetate buffer brings the once shrunken gel back to a swelled state again. Production of cross-linking points by a cyclic ester linkage between the phenylboronic acid group and the ribose unit of AMP and the electrostatic interaction of the anionic phosphate group of AMP with the cationic ammonium groups as well as neutralization of ionizable groups contributes to shrinkage of the gel layer. Thus, the cis-diol recognition ability of boronic acids is implemented effectively to the nucleotide-responsive AFM tip.

Totally synthetic polymer with lectin-like function: Induction of killer cells by the copolymer of 3-acrylamidophenylboronic acid withN,N-dimethylacrylamide

Lectin-like function was demonstrated in this study for a novel water-soluble polymer with phenylboronic acid residues (poly (AAPBA-DMAm)), which induced appreciable proliferation of murine spleen lymphocytes with an increased expression of interleukin-2 (IL-2) receptor on their surface. Consequently, boosted proliferation of lymphocytes with cytotoxic action to YAC-1 cells was achieved by concurrent addition of IL-2 with poly(AAPBA-DMAm) in the medium, indicating this boronate-containing polymer to be worked as an effective immuno-adjuvant for the induction of lymphokine-activated killer (LAK) cells. Flow-cytofluorimetry study revealed that poly(AAPBA-DMAm) competitively inhibited the cellular binding of N-acetylneuraminic acid-specific lectin (Limax Flavus Agglutinin) in a concentration-dependent manner, suggesting that phenylboronate moiety in the polymer may recognize N-acetylneur- aminic acid (sialic acid) residues existing on the plasma-membrane surface of lymphocytes to induce their proliferation.

Separation Behavior of Biological Constituents Having cis-Diol Groups through Interactions with Phenylboronic Acid Sites Introduced on the Inner Wall of a Fused-Silica Capillary

Abstract Phenylboronic acid sites were introduced onto the inner wall of a fused-silica capillary through the polymerization of 3-acrylamidophenylboronic acid after activating the wall by a silane coupling reagent. The phenylboronic acid-modified capillary was examined from various viewpoints of separation chemistry. Nucleosides having cis-diol groups were mainly used as model samples. The phenylboronic acid-modified capillary successfully separated a mixture of nucleosides, for example, an adenosine and deoxyadenosine mixture, in capillary electrophoresis. The separation was based on an interaction between the cis-diol groups and the phenylboronic acid sites on the capillary wall. The cooperation of borate migration buffer with the modified capillary could improve the resolution. The separation of a d- and l-glucoses mixture was also indicated with the coexistence of β-cyclodextrin in electrophoresis using a modified capillary. Furthermore, mixtures of nucleosides were separated with satisfactory results by chromatography utilizing a solute-wall interaction in the modified capillary, where the eluate was delivered through the capillary by a gravity method without any electrophoresis. The separation of biological constituents in this study, which took advantage of the interaction between the solutes and the inner wall of the capillary, is interesting as a new separation technique in not only capillary electrophoresis, but also capillary chromatography.

Interfacial Recognition of Sugars by Novel Boronic Acid-Carrying Amphiphiles Prepared with a Lipophilic Radical Initiator

Novel amphiphiles which carry several boronic acid groups in their polar head regions were prepared by copolymerization of 3-(acrylamido)phenylboronic acid (APBA) and ((N,N-dimethylamino)propyl)methacrylamide (DMAPMA) using a lipophilic radical initiator. The amphiphiles obtained (Lipid AD) formed a stable monolayer, and the surface pressure−molecular area (π−A) profile of the monolayer was changed by the addition of sugars in the subphase. The effect was dependent on the structure of the sugars added, which is consistent with the effect of the sugars on the viscosity of aqueous solution of the copolymer of APBA and DMAPMA. The presence of DMAPMA residues together with the APBA residues in the head group of the amphiphile was more effective on the recognition of sugars than that of acrylamide or (3-(methacryloylamino)propyl)trimethylammonium chloride residues, which confirmed the supporting effect of tertiary amines on the recognition of sugars by phenylboronic acid groups previously reported. The monolay...

Effect of phenylboronic acid groups in copolymers on endothelial cell differentiation into capillary structures

—Copolymers (IABb) composed of N-isopropylacrylamide (I), N-(3-dimethylaminopropyl)-acrylamide (A), 3-acrylamidophenylboronic acid (B), and a hydrophobic comonomer, n-butyl methacrylate (b), were synthesized as cell culture substrata since we previously learned that bovine aortic endothelial cells (BAECs) cultured on the phenylboronic acid-containing copolymer (IAB) differentiated into capillary structures after 26 days culture. The synthesized IABb copolymers contained higher boron and amine moieties than the IAB copolymer. The results of the dynamic contact angle measurement revealed that IABb copolymer-coated surfaces showed a relatively hydrophobic nature, changing to hydrophilic in response to the aqueous environment. BAECs cultured on the copolymer substrata developed into capillary networks after 7 days. This is probably due to the enrichment of boron and amine segments in the vicinity of the hydrophilic copolymer surface, enhancing more pronounced interaction of boronates with cell membrane glycocalyx. The introduction of n-butyl methacrylate into the polymers might enhance the diffusion of the hydrophobic segments to the bulk polymers and the concentration of relatively hydrophilic segments at the outermost polymer surfaces by contact with water. A copolymer (IAP) without boronic acid groups was also prepared using N-phenylacrylamide (P) as a comonomer instead of 3-acrylamidophenylboronic acid in the IAB copolymer to investigate the effect of boronic acid moieties on the capillary formation of the cultured cells. Although the endothelial cells seeded on the copolymer without boronic acid groups adhered during the early culture period, these cells showed neither proliferation nor differentiation and detached from the surface after 13 days. These results strongly support the opinion that the phenylboronic acid groups in the copolymers are responsible for the specific induction of tissue formation of BAECs through the interaction with glycoconjugates on the cell membranes.

Boronate-Containing Polymer as Novel Mitogen for Lymphocytes

A novel water-soluble polymer (poly(AAm-co-PBA)) was synthesized by radical copolymerization of 3-acrylamidophenylboronic acid with acrylamide and evaluated as synthetic mitogen of lymphocytes. Boronic acid was introduced in the polymer as recognition site of sugar residues existing on the plasma membrane surface of lymphocyte, since it makes a covalent bonding with polyol compounds including sugars. Obvious increase in [ 3H]thymidine incorporation of lymphocytes was observed when murine spleen lymphocytes were cultured with poly(AAm-co-PBA), while little proliferative response of lymphocytes was observed in the presence of the homopolymer of acrylamide, the polymer without boronic acid moiety. This suggests that proliferation of lymphocytes was induced through the binding of phenylboronic acid moiety in poly(AAm-co-PBA) with glycoproteins existing on the plasma membrane surface of lymphocytes. Further, poly(AAm-co-PBA) was found to induce even the proliferation of B cell-depleted population of lymphocytes, suggesting T cell stimulation by this boronate-containing polymer.