Biosensors Based on Entrapment of Enzymes in a Water-Dispersed

et Appliquée, Département de Chimie et Biochimie, Université du Québec à Montréal, C.P. 8888, Succursale A, Montreal, Quebec H3C 3P8, Canada...
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Chapter 3

Biosensors Based on Entrapment of Enzymes in a Water-Dispersed Anionic Polymer

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch003

Guy Fortier, Jian Wei Chen, and Daniel Bélanger Groupe de Recherche en Enzymologie Fondamentale et Appliquée, Département de Chimie et Biochimie, Université du Québec à Montréal, C.P. 8888, Succursale A, Montreal, Quebec H 3 C 3P8, Canada

Biosensors using choline and glucose oxidase have been prepared by deposition at the surface of platinum electrodes of an aliquot containing a blend of amorphous polyester cationic exchangers (AQ 29D:AQ 55D; ratio 1:1) dispersed in water and the enzyme. The polymer-enzyme films were prepared according to the following three protocols: 1) the enzymatic film is dried at room temperature, 2) same as 1 and covered by a Nafion overlayer or 3) the enzymatic film is dried by heating at 50°C for 30 min. The evaluation of the biosensors was based on the amperometric detection of enzymatically generated hydrogen peroxide in presence of choline chloride or glucose. Enzyme electrodes prepared using protocols 2 & 3 are stable but those dried by heating at 50°C for 30 min gave the higher amperometric responses. Also, the AQ-enzymatic film keeps the same permselectivity against small anionic redox species than the A Q film alone of the same thickness.

During the last decade, immobilization of oxidase type enzymes by physical entrapment in conducting or ionic polymers has gained in interest, particularly in the biosensor field. This was related to the possibility for direct electron tranfer between the redox enzyme and the electroconducting polymers such as polypyrrole (1,2), poly-N-methyl pyrrole (3), polyindole (4) and polyaniline (5) or by the possibility to incorporate by ion-exchange in polymer such as Nafion (6) soluble redox mediators that can act as electron shuttle between the enzyme and the electrode. Previously, Nafion was used in electroanalysis as electrode covering membrane to immobilize cationic species (7,8) and as a barrier to anionic species (9,10). In biosensor field, the main limitation in the use of Nafion for enzyme entrapment is related to the solubility of the Nafion in lower aliphatic alcohols which are not compatible with the enzyme activity (11). Until now, only glucose oxidase was successfully immobilized in Nafion.

0097-6156/92/0487-O022$06.O0A) © 1992 American Chemical Society

In Biosensors and Chemical Sensors; Edelman, Peter G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch003

3. FORTIER ET AL.

23

Biosensors Based on Entrapment of Enzymes

More recently, a new series of water dispersed anionic polymers, the A Q 29D, 38D and 55D polymers were released by Eastman Kodak. Since that time, these polymers were used as electrode modifier (72, 13), as covering membrane (14) and as support for enzyme immobilization (15, 16). A Q polymers are high molecular weights (14,000 to 16,000 Da) sulfonated polyester type polymers ( 17, 18). Their possible structures have been recently presented (18). The A Q polymer série shows many interesting characteristics useful for the fabrication of biosensors. They are water dispersed polymers and thus compatible with enzymatic activity. They have sulfonated pendant groups similar to Nafion and they can act as a membrane barrier for anionic interferring substances and they offer the possibility to immobilize redox mediators by ion exchange. In this paper, we have evaluated three different protocols for the preparation of AQ-enzyme film using choline and glucose oxidases and mixture of A Q 29D and A Q 55D (1:1). This A Q mixture is recommended by Eastman Kodak to increase the adherency of the film to a surface such as a platinum electrode (17). The values 29 and 55 represent the glass transition temperature of each polymer. Also, the main structural difference between the two polymers is that, in the case of A Q 55D, an aliphatic glycol moiety replaces the cycloaliphatic glycol moiety found in the AQ29 (17,18). These biosensors transform their respective substrates following the reactions 1 and 2. Choline

+ 0

2

Cholinç oxidase >

Betaine aldehyde + 0

2

Choline oxidase > Betaine

β-d-Glucose

+ 0

2

G

l u c

c

° S oxidase

>

Bet

a i n e aldehyde + H °2 2

+

la

(lb)

d-Gluconolactone+ H °2 2

2

()

Material and Methods Reagents. Choline oxidase and glucose oxidase and their respective substrates, choline chloride and glucose were purchased from Sigma Chemical Co. (StLouis, USA). The cationic exchangers A Q 29D and 55D were kindly supplied by Eastman Chemical Inc. (Kingsport, USA) and were obtained as dispersed polymer solutions at concentration of 30 and 28% (w/v) in water, respectively. A blend of the A Q polymer solutions was prepared by mixing the A Q 29D with the A Q 55D in a ratio (1:1), and was further diluted with water to a final concentration % (w/v) indicated in the text. Nafion (equivalent mass 1100 g) 5% (w/v) in a mixture of lower aliphatic alcohols and 10% of water was obtained from Aldrich (St.Louis, USA) and diluted with methanol to yield a stock solution of 0.5% (w/v). 2

F i l m Fabrication. The platinum electrode (0.28 c m area) was fabricated and cleaned as previously described (19). Thin films of AQ-enzyme were prepared by dissolving an amount of the enzyme, as indicated below, in 10 μΐ of 1.5% A Q polymers solution at room temperature. Two aliquots of 5 μΐ were deposited atop the platinum electrode and the first aliquot was allowed to dry before the second addition. This procedure corresponds to the first protocol. In addition, for the second protocol, 10 μΐ of the 0.5% Nafion solution was casted atop the dried AQ-enzyme film and the methanol was allowed to evaporate at room temperature. The third protocol consisted in the deposition of 10 μΐ of a 1% of A Q solution containing the enzyme, atop the platinum electrode followed by heating in an oven at 50°C during 30 min. In each case, 2 U of glucose oxidase were used.

In Biosensors and Chemical Sensors; Edelman, Peter G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

24

BIOSENSORS AND CHEMICAL SENSORS

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch003

A l l the enzyme electrodes were held at 0.7 V vs S C E in 5 ml of 0.1 M phosphate buffer, pH 7, for one minute before addition of their respective substrate. The steady-state values recorded after 1 min were used to obtain the calibration curves. The electrode was removed from the solution and washed with water prior to be used for another assay. Instrumentation. A l l electrochemical experiments were carried out in a conventional one-compartment cell. Potentials were applied to the cell with a bipotentiostat (Pine Instruments Inc., USA) model RDE4. Current-time responses were recorded on a X Y Y ' recorder model B D 91 (Kipp & Zonen, USA) equiped with a time base module. A l l potentials were measured and quoted against a saturated calomel electrode (SCE). K i n e t i c A n a l y s i s . The kinetic parameters were obtained by iterative non-linear curve fitting of raw data (current generated versus the substrate concentration).The data fitted a modified Michaelis-Menten equation: Is = (Imax [S] )/ (Km' ' + [S])

(3)

where Is is the observed current and Imax is the maximal value for the current response. K m " is an effective apparent constant that represents the substrate concentration giving one half of the Imax in air saturated solution. The K m " is compounded by the mass transport rates of both substrates because all the currents used were obtained at steady-state. Also, we have made the assumption that the structure of the enzyme is not modified following the immobilization. Results and Discussion Three different protocols have been evaluated for the enzyme-AQ film preparation to obtain the enzymatic film that will be the most stable in the assay solution. These protocols consisted i) in depositing the mixture of glucose oxidase and A Q atop the platinum electrode and allowing the solvent to evaporate at room temperature, ii) in covering this AQ-glucose oxidase film with a Nafion layer or iii) in heating the AQglucose oxidase film at 50°C in an oven during 30 min. The reproducibility of the time-current traces for 4 consecutive assays of 20 m M of glucose was evaluated as probe of the film stability for each protocol. Figure 1 depicts the stability of the glucose oxidase-AQ film obtained following the different protocols. When the glucose biosensor is prepared by mixing 10 μΐ of A Q 1.5% mixture with 2 U of glucose oxidase (Fig.lA), the value of the steady-state current decreases by 25% after each glucose assay. For the 5th assay, no response was observed indicating that the film has been slowly dissolved in the assay solution during each assay. To aleviate the dissolution of the enzymatic film during the glucose assay procedure, a covering membrane made by addition of 10 μΐ of Nafion 0.5% was used. The amperometric current increases until a constant value of 20 μΑ is reached on the fourth glucose assay (Fig. IB). The addition of Nafion film atop of the AQenzyme layer doesn't seem to denaturate the enzyme entrapped in the A Q film because the current reached a similar value as the current observed in absence of Nafion. On the other hand, the use of Nafion as covering membrane gives a thicker film that leads to mass transport limitation of the substrates in the A Q film (20). It results in a longer time to obtain the steady-state current. The last protocol, similar to the one use by Wang and co-workers (16), consisted in heating the enzymatic film in an oven at 50°C for 30 min. After this period, a glassy and resistant enzymatic film is obtained. An enzyme loading of 2 U

In Biosensors and Chemical Sensors; Edelman, Peter G., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 3, 2016 | http://pubs.acs.org Publication Date: April 23, 1992 | doi: 10.1021/bk-1992-0487.ch003

3. FORTIER ET A L

Biosensors Based on Entrapment of Enzymes