Yasuo Yoshimi

`Gate effect' of thin layer of molecularly-imprinted poly(methacrylic acid-co-ethyleneglycol dimethacrylate)

Abstract Molecular imprinting is a new technology for the synthesis of polymers with antibody-like specificity, which are potentially useful in sensing or separation devices. Here, we report that a thin layer of molecularly-imprinted copolymer exhibits an increase in its diffusive permeability in the presence of its template. A thin layer of molecularly-imprinted poly(methacrylic acid-co-ethyleneglycol dimethacrylate) of theophylline was observed to form on the surface of electrically conductive indium-tin oxide (ITO) film. Cyclic voltammetry of ferrocyanide was performed using the copolymer-grafted ITO as a working electrode in the presence and absence of the template. The presence of the template was found to enhance the anodic current remarkably, which suggests that the diffusive permeability of the thin layer of the molecularly-imprinted polymer (MIP) is sensitive to the presence of its template molecule. This idea is supported by atomic force microscopy (AFM) in which the surface porosity of the layer of the MIP was observed to increased in the presence of its template. We conclude that the structure and diffusive permeability of a thin layer of molecularly-imprinted poly(methacrylic acid-co-ethyleneglycol dimethacrylate) enables this system to be used as a sensor for a given template.

Detection of deposition rate of plasma-polymerized films by quartz crystal microbalance

Abstract The deposition rates of plasma-polymerized (pp-) films of styrene, pentafluorostyrene, allyl alcohol, allylamine, methacrolein, acrolein, and acrylic acid were determined by the quartz crystal microbalance technique. Using the same polymerization conditions (100 W RF and 100 Pa vapor pressure) for the various monomers, it was found that the deposition rates were proportional to the polymerization time. The deposition rates of pp-styrene, pp-pentafluorostyrene, pp-allyl alcohol and pp-allylamine were independent of the position of the lower electrode in the plasma polymerization equipment. In contrast, the deposition rates of pp-methacrolein, pp-acrolein and pp-acrylic acid were scattered. The average deposition rate of pp-allyl alcohol was 0.42 μg/min. The average deposition rate of pp-styrene was higher by eight times, and that of pp-pentafluorostyrene was higher by 388 times than that of pp-allyl alcohol. The average deposition rate depended on the chemical structure of the monomer, giving rise to different mechanisms of polymerization. The deposition rates of monomers containing allylic hydrogens were slow due to radical–radical coupling. These unexpected but significant differences were assumed to have arisen from initiation-controlled free radical polymerization.

Turning of contact angle on glass plates coated with plasma-polymerized styrene, allylamine and acrylic acid

Plasma polymerization of styrene, allylamine and acrylic acid was performed under nine polymerization conditions. The obtained polymers generated the film-state only under these conditions. Over 90 days, the contact angles of plasma-polymerized (pp)-films were measured on glass plates coated with pp-styrene, pp-allylamine and pp-acrylic acid. The values of the contact angles of pp-styrene, pp-allylamine and pp-acrylic acid were scattered at the initial stage under the nine polymerization conditions. However, the contact angles of these films after 40 days of storage under vacuum condition always converged into one value. These converged values of contact angles were about 87° for pp-styrene, 67° for pp-allylamine and 28° for pp-acrylic acid. We assumed that this environmental adaptability of contact angle came from the turnover of the polar functional groups between the polymer surface and bulk.

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.

Surface modifications of functional electrodes of a light addressable potentiometric sensor (LAPS): non-dependency of pH sensitivity on the surface functional group

Abstract To achieve a functional surface design on the electrode of a light addressable Potentiometric sensor (LAPS), two different surface modification techniques were adopted to determine the effect of surface functional groups on the sensitivity and reliability of the LAPS. One was chemisorption of alkanethiols (HS-(CH 2 ) n -X, n is 2 or 11, X is CH 3 , COOH or NH 2 ) on a gold-deposited electrode, and the other was photochemical immobilization of copolymers containing benzophenone and functional groups of CON(CH 3 ) 2 , COOH and N(CH 3 ) 2 in their side chains on a propyltrimethoxysilane-treated electrode. Linearity was observed between the potentials of inflection points on photocurrent-potential curves ( V pip ) and the pH of the contacting solution: V pip = a – b · pH. Gold deposition on the electrode reduced both the pH sensitivity of the V pip and the degree of linearity of relationship between V pip and pH. The alkanethiol chemisorption on the gold surface hardly changed the V pip -pH relationship irrespective of the terminal functional groups of the alkanethiols. Neither the propyltrimethoxysilane treatment nor the photogel immobilizations changed the pH sensitivities of the V pip or the linearities between V pip and at pH 3–8. In conclusion, gold deposition is not suitable for modification of the LAPS electrode, while surface modification with photogel immobilization of a propyltrimethoxysilane-treated electrode can be used without reducing the pH sensitivity or reliability.

Development of an artificial synapse using an electrochemical micropump

Improving the resolution of artificial sensory organs requires an interface that receives external information from electronic circuits and stimulates appropriate neurons individually in response to that information. The method of electric stimulation in available artificial sensory organs is fairly nonselective; therefore, we developed a method of chemical stimulation of neurons using a neurotransmitter containing an electrochemical micropump powered by the bubbling that occurs during water electrolysis. The micropump contains a glass nozzle with a tip 10 µm in diameter. Two blackened platinum electrodes for the electrolysis were inserted into the body of the pump, which was filled with neurotransmitter solution. The distance between a neuron of the gastropod Aplysia and the tip of the nozzle was adjusted to about 100 µm. A potential difference of 3.0 V was applied to the electrodes to propel the solution toward the neuron while its membrane potential was monitored. Administration of 1-mM acetylcholine to a resting neuron caused neural firing only when the voltage was applied for 0.5 s and without a time lag. During administration of 50-mM γ-aminobutyric acid to spontaneously firing neurons, the firing disappeared with a time lag of 1 s after application of 3.0 V. We concluded that an electrochemical micropump can be applied for rapid neurotransmitter administration to control the excitation and inhibition of neurons. This simple pump can be miniaturized to create “synapses” in artificial sensory organs.

Development of a glucose sensor with on/off control of enzyme activity without the effects of protein adsorption

The main factor mitigating against the realization of a hypodermically inserted glucose sensor for an artificial pancreas is the change in response current due to fibroblast adhesion and protein adsorption to the sensor surface. To overcome this problem, we have developed a method whereby the activity of glucose oxidase (GOD) fixed on the membrane of the sensor surface is switched on and off, and measurements are made during a transient state in which the glucose concentration gradient within the GOD membrane is small. Measuring in a transient state while GOD activity is being controlled, a correlation was observed between glucose concentration and response current in a phosphate buffer solution. Calibration curves of response current against glucose concentration in aqueous solutions of human serum albumin and in phosphate buffer solution were then compared using the transient method and a steady state method without control of GOD activity. In addition, glucose concentration was measured in bovine plasma for 480 min, and the time courses of the response currents for the transient and steady state measurements were compared. It was found that in both experiments the response current decreased greatly under steady state measurement as a result of protein adsorption, but during the transient measurement, response current was virtually unchanged. By measuring glucose concentration in the transient state while controlling GOD activity, it is possible to inhibit the effects of protein adsorption. ASAIO Journal 1997; 43:M505-M509.

Improved gate effect enantioselectivity of phenylalanine-imprinted polymers in water by blending crosslinkers.

In this work, the anodic current at an electrode grafted with a molecularly imprinted polymer (MIP) crosslinked via a combination of hydrophobic ethyleneglycol dimethacrylate (EDMA) and hydrophilic methylene bisacrylamide (MBAA) was found to exhibit enantioselective sensitivity to the phenylalanine template in aqueous solution. An MIP-grafted electrode crosslinked with a 2:1 mixture of EDMA and MBAA was observed to respond to the template with the highest enantioselectivity, such that the change in current induced by the imprinted template was more than four times that induced by the enantiomer of the template. The contact angle of a water droplet on an MIP-coated electrode prepared using the optimal crosslinker blending ratio was also sensitive to the template and again exhibited chiral selectivity. The change in the contact angle induced by the template was more than twice as large as that obtained from the templates enantiomer. Atomic force microscopy showed that the surface of the MIP layer fabricated using a mixture of crosslinkers was rougher than that made with a single crosslinking agent, although there was no apparent correlation between the roughness and the enantioselectivity of the layer. These results indicate that the appropriate combination of crosslinkers enables the chiral-selective gate effect by modulating the flexibility and hydrophilicity of the MIP layer. The approach described herein therefore represents a new means of improving the selectivity of MIPs by blending crosslinkers having different chemical properties.

A new reagentless immunosensor for measuring IgG concentration in human plasma based on fluorescence-enhancement immunoassay

Abstract Based on the enhancement of fluorescein isothiocyanate (FITC) fluorescence caused by binding to proteins, we developed a reagentless immunosensor to directly determine immunoglobulin G (IgG) in human plasma. The fluorescence-enhancement immunoassay (FEI) is used in only limited conditions in spite of its simplicity, because the homogeneous reagent cannot be reused. In the present study, a new FEI was evaluated with an immobilized reagent. FITC was used to label protein A, which has specific reactivity with IgG. The FITC-labeled protein A was immobilized on nylon membrane and placed in a fluorometer cell. It produced fluorescence enhancement corresponding to IgG concentration in phosphate-buffered saline (PBS) and bovine serum solution, and it responded to IgG also in human plasma. Thus the IgG concentration can be determined directly using the reagentless immunosensor based on FEI.

Changes in the Porosity and Permeability of a Molecularly Imprinted Membrane Induced by the Adsorption of a Trace Quantity of Template

It is known that the diffusive permeability of solutes within a thin layer of molecularly imprinted polymer (MIP) may be affected by specific binding of the MIP with its template molecule. This phenomenon, termed the gate effect, shows promise for the development of novel biomimetic sensors. However, the mechanism underlying this effect is still unclear; although the relationship between the specific adsorption of a template and the corresponding porosity and permeability of the polymeric film or membrane is very important, this association has not yet been examined in detail. We therefore studied this relationship using a molecularly imprinted self-supporting membrane (MISSM) possessing chiral specificity, specially developed as a tool for investigating the gate effect. Both the diffusive permeability and volume porosity of the MISSM were sensitive to the presence of the template compounds (D and L-phenylalanine) at concentrations as low as 5 µM, while, at the same time, insensitive to the enantiomer of the template. The relationship between the amount of adsorbed template and the equilibrium template concentration followed the expected Langmuir isotherm pattern, which indicates the thermodynamic homogeneity of binding sites in the MISSM. This study also demonstrated that the relative concentration of the adsorbed template in the membrane was only 3 ppm and relative site occupation was only 1% following exposure to a 5 µM concentration of the template. These results show that the gate effect may be advantageously exploited during application of MIPs in amplifiers or sensors offering high sensitivity. Molecularly imprinted polymers (MIPs) are synthetic polymers that contain specific binding sites formed by imprinting of a target molecule (or template) during the polymerization process. An MIP layer can be prepared by a simple and economical procedure (1-3). Before such MIPs may be employed as molecular recognition elements in chemical-sensing devices, however, it is necessary to develop a means of translating specific binding events at the MIP into an electric signal. The so-called gate effect, which refers to changes in the diffusive permeability of solutes within the MIP layer resulting from specific binding at the template, can be used as a mechanism for signal transference based on conductometry, utilizing changes in the ionic permeability of the MIP membrane associated with a specific interaction with the template (4-6). In this gate analogy, the template corresponds to the key while the MIP site which allows specific rebinding with the template corresponds to the keyhole. An amperometric method is also applicable, using a thin MIP layer grafted onto an electrode, in which the template can be detected by following changes in the faradic current resulting from the change in permeability of a redox marker across the MIP layer (7-11). The gate effect is a selective process capable of discriminating between