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Use of porous ion exchange film for glucose sensing.


The ability to monitor a low level of various compounds is crucial in environmental science and in clinical practice. Objectives of biosensor development include portability, high detection sensitivity (i.e., at least to the micromolar level), rapid detection, and accuracy of detection. Moreover, sensor devices are desired to be produced at a low cost. Conventional biosensors, such as ion-sensitive field effect transistors, which enable detection of amino acids, proteins, and DNA are two-dimensional systems (1), (2). Other biosensors are designed based on the electrochemical impedance spectroscopy (3), (4). In this study, we focused on a recently developed porous ion exchange resin (5-7) because the open-cellular three-dimensional (3D) structure of the resin film can be exploited for sensitive detection of target compounds. We have successfully detected both cations ([Ca.sup.2]) and anions ([Cl.sup.-)] using the film (8).

High performance detection of biological materials, which typically involves an enzyme catalyzed conversion of the target compound to a detectable product, requires the preservation of the biological activity of the target compound. Typically, enzymes are covalently bonded to a support; however, this bond can disrupt enzyme activity. In contrast, enzymatic activity is preserved in an ionic bond. In this study, we utilize a porous monolith-type ion exchanger to explore the detection of glucose as a relevant representative of the target compound.


Porous Monolith-Type Ion Exchanger

The 3D, open-cellular ion exchange resin film is depicted in Fig. 1. The resin formed using styrene and divinylbenzene constituted the framework of the monolith. The porous monolith ion exchanger was prepared by polymerization of water-in-oil emulsions without stirring as described in Reference 7. In this study, we used the cation exchange film contained the sulfonic acid groups and the anion exchange film contained the quaternary ammonium groups. Figure 2 show the diagrams for ion exchange on each films. Ion exchange functional groups, such as sulfonic acid groups for cation exchange and quaternary ammonium groups for anion exchange, were distributed both on the resin surface and throughout the polymer.



The resin mesopores were created by applying water drops during emulsion polymerization. The pores with various sizes (5-50 [micro]) accommodate small molecules such as glucose, as well as larger compounds such as glucose oxidase (GOD), proteins, DNA, and other biopolymers, throughout the monolith rapidly and homogeneously.

In general, the ion exchanger adsorbs counter ions of the ion exchange group. Ion makes the impedance of the ion exchanger increase due to suppression of ion conduction between the functional groups as a result of the large interaction strength compared with proton conduction.

Selection of GOD as Test Enzyme

GOD was selected as a representative enzyme in this study. This enzyme contains amino and carboxyl groups (isoelectric pH of 4.5), which allow pH-mediated charge adaptation. In addition, GOD catalyzes the oxidation of glucose--a compound whose rapid and accurate detection is clinically relevant in testing for diabetes. GOD is a globular protein with an approximate molecular weight of 150 kDa and a maximum diameter of several dozen nanometer. Therefore, it would be easily accommodated within the resin structure. Because the spacing between ion exchange groups on the porous ion exchange film was less than 1 nm, immobilization of GOD would be achieved by many ion exchange groups. This would result in a strong GOD--resin bond though an individual ionic bonds has a relatively weak bonding force.

GOD Impedance Measurements

Impedance measurements were performed using instrumentation and ZVIEW[TM] software obtained from Solartron Public Company (Bankok, Thailand). The ion exchanger was quarried out cylindrically. The diameter and length of the sample were 10 and 4 mm, respectively. The exchanging capacity of the porous monolith-type ion exchanger was 0.22 mmol/ml. Therefore, the exchanging capacity of the sample 4 - mm in length was 0.08 mmol. The resin was positioned in a flow tube. The electrode was made of 8-mm diameter platinum mesh, which contacts both sides of the ion exchanger. The measurement cell was keeped up vertically to exclude air from the cell during bottom-to-top flow of the measuring solution. The measuring solution was maintained at 25[degrees]C using a heater and was passed into the column using a tube pump with a flux of 2.8 ml/min. Impedance measurements were performed using a 10-mV AC signal at frequencies from 100 mHz to 10 kHz.

The porous cation exchanger was prepared by [H.sup.+] substitution using 1 mol/ml HCl followed by rinsing with deionized water. Preparation of the anion exchanger involved [OH.sup.-] substitution using 1 mol/ml NaOH. A GOD solution was poured into the column followed by rinsing with deionized water. Impedance measurements were performed for the above-prepared ion exchange films and films exposed to glucose.


Modification of GOD

We measured the impedance of the porous ion exchange film after treatment with 10 ml of 6.7 X [10.sup.-5] mol/1 GOD. Figure 3 shows the frequency dependence of the impedance |Z| for GOD. In the impedance-frequency ([absolute value of Z]-f) characteristics of the anion exchangers, the increase of [absolute value of Z] was observed in the low frequency range. This is possibly due to the influence of the electric double layer which is formed on the electrode surface. On the other hand, difference of the impedance between [OH.sup.-] and GOD was observed in the high frequency range. The impedance of GOD immobilized film is higher than that of [OH.sup.-] substituted film at 10kHz. It is indicated that the immobilization of GOD opposes proton conduction of the exchange film.


Impedance nearly doubles for both cation and anion films (Table 1), indicating that GOD efficiently substitutes for [H.sup.+] and [OH.sup.-] due to the presence of [COO.sup.-] and [NH.sub.4.sup.+] residues. Cation exchange film absorbed [H.sup.+] shows the acidic property, and its pH is lower than isoelectric point (pH 4.35). As arginine, histidine, and lycine in GOD have positive charge, GOD is immobilized on the resin. In contrast, pH of anion exchange film is higher than isoelectric point. As glutamic and asparagine acids have negative charge, GOD is immobilized on the anoin film. GOD adsorption was confirmed due to UV irradiation. The benzene ring in GOD absorbs UV energy; preliminary standard curve experiments established that the absorption was proportional to enzyme concentration (data not shown). We observed that the UV absorbance of the enzyme solution decreased after passage through the resin, indicating adsorption due to GOD over the resin (estimated at 1.0 X [10.sup.-7] mol, based on comparison with standard curve). In addition, the resin impedance increased simultaneously.
TABLE 1. [absolute value of Z] change at 10
kHz by enzyme modification.

 [absolute value of Z]([OHM])

 Initial GOD

Anion exchanger 75.805 135.43
Cation exchanger 51.128 99.414

Glucose-Mediated Impedance

When resin containing adsorbed GOD was exposed to the flow of a glucose solution, the impedance of the anion exchange film increased, whereas that of the cation exchange film exhibited little change (see Fig. 4). This might be possibly due to the production and subsequent adsorption of another anion. Decomposition of glucose by the resin-bound GOD was evident by the detection of gluconic acid and hydrogen peroxide (see Fig. 5). A substantial impedance change was produced by gluconic acid for the anionic films (Table 2).

TABLE 2. [absolute value of Z] change at
10 kHz of solutions in anion exchange films.

 [absolute value of Z]([OHM])

Initial 73.231
Glucose 72.829
Gluconic acid 300.49
Hydrogen peroxide 74.300

We quantified the reacting amount of glucose with the phenol-sulfuric acid method. Table 3 shows the absorbance for 490-nm wavelength and concentration of the glucose solutions which are before and after contacting with resin-adsorbed GOD. The data of absorbance for 490-nm wavelength in Table 3 shows the value of the diluted solutions (1%), however the data of concentration show the value of solution without dilution. It is supposed that the glucose of 3.4 X [10.sup.-5] mol reacted with the enzyme. This result indicates that the glucose of about 25% was reacted, while gluconic acid was produced with GOD and glucose. The optimal pH range of GOD is pH 3-6.5. It is supposed that its enzymatic activity was not best, because the surface of anion exchange film absorbed GOD bias a basic.
TABLE 3. Absorbance for 490 nm wavelength and
concentration of glucose solution.

 Absorbance (490 nm) Concentration
 (X [10.sup.-2]mol/l)

Before passing 0.38 1.44
After passing 0.29 1.10

Influence of Glucose Concentration on Impedance

We examined the detection of various concentrations of glucose (in the form of gluconic acid) on anionic films. Figure 6 shows the impedance modulus ([absolute value of Z]) at 10 kHz plotted as a function of glucose concentration. The linear relationship between [absolute value of Z] increase and glucose concentration was indicated.



We have demonstrated that GOD can be effectively fixed throughout the 3D network of a porous open-cellular monolith resin with a pore diameter of 5-50 [micro]m. The gluconic acid produced by the decomposition of glucose due to immobilized GOD, is detectable, leading to an increase in the impedance of the anion exchange film. The impedance increase is proportional to (glucose levels) the concentration of the gluconic acid. Because this sensing method uses the enzyme original activity, it is probable that not only the glucose but also other biological materials can be detected by using the porous ion exchanger. In addition, the sensing method by using the ion exchanger is possible to keep high activity of the biological materials. There is a high potentiality that the porous ion exchanger is applicable to a high performance device for biological sensing.


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(2.) M. Castellarnau, N. Zine, J. Bausells, C. Madrid, A. Juarez, J. Samitier, and A. Errachid, Sens. Actuators B, 120, 615 (2007).

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(8.) H. Aoki, K. Miyano, D. Yano, K. Sano, K, Yamanaka, C. Kimura and T. Sugino, Polym. Eng. Set., DOI 10.1002/pen (2007).

Saori Hotta, (1) Kazuki Miyano, (1) Hidemitsu Aoki, (1) Nobuaki Fujiwara, (2) Akihiko Masui, (2) Daisaku Yano, (3) Kazuhiko Sano, (3) Koji Yamanaka, (3) Chiharu Kimura, (1) Takashi Sugino (1)

(1) Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka, 565-0871 Japan

(2) Technology Research Institute of Osaka Prefecture, 2-7-1 Ayumino, Izumi, Osaka, 594-1157 Japan

(3) Organo Corporation, 4-4-1 Nishionuma, Sagamihara, Kanagawa, 229-0012 Japan

Correspondence to: Dr. Hidemitsu Aoki; e-mail:

DOI 10.1002/pen.21288

Published online in Wiley InterScience (

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Article Details
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Author:Hotta, Saori; Miyano, Kazuki; Aoki, Hidemitsu; Fujiwara, Nobuaki; Masui, Akihiko; Yano, Daisaku; San
Publication:Polymer Engineering and Science
Article Type:Report
Geographic Code:9JAPA
Date:May 1, 2009
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