4.1. Fabrication of Glucose Biosensor

4.1.1. Glucose Biosensor

Use H2O2 Sensor for Transducer. Glucose sensor can be made in many different ways by using the enzyme glucose oxidase. This enzyme glucose oxidase catalizes the following reaction:

To measure the glucose concentration, three different methods can be used:

1. Measure oxygen consumption by an oxygen sensor

2. Measure acid (gluconic acid) production by a pH sensor

3. Measure production of H2O2 by a peroxide sensor.

Note that an oxygen sensor is a transducer that converts oxygen concentration into electrical current. A pH sensor is a transducer that converts pH change into voltage change. Similarly, a peroxidase sensor is a transducer that converts peroxidase concentration into an electrical current.

Structure of Biosensor. The structure of the biosensor to be made is shown in Fig. 4.1. The base transducer consists of H2O2 sensor which is essentially the same as the oxygen sensor fabricated in Section 2.2 (the signal conditioning circuit is shown in Fig. 2.11). The enzyme glucose oxidase is immobilized in front of the H2O2 sensor between two membranes. The inner membrane is a permeselective membrane that allows passage of H2O2 where as the outer membrane separates the biosensor from measurement medium.

Enzyme Immobilization. The enzyme can be immobilized by: (1) physical entrapment; (2) cross-linking; and (3) covalent attachment. All three methods are to be used. The enzyme used is glucose oxidase in powder form (Sigma Type II, specific activity 25 IU/mg - see Section 3.2.2 for the definition of specific activity).

4.1.2. Glucose Biosensor by Physical Adsorption

Step 1. Prepare a mixture of 24 g of cyclohexanone, 24 g of acetone and 1 g of cellulose acetate (39.8% acetyl content, available from Aldrich). This is for casting the permeselective membrane.

Fig. 4.1. Overall structure of glucose biosensor.

Fig. 4.2. Glucose biosensor by physical adsorption of enzyme.

Step 2. Stir the mixture at room temperature until the cellulose acetate has dissolved and then cast a thin film on to the surface of the sensor probe. Allow the solvent to evaporate.

Step 3. Dissolve glucose oxidase in 0.1 M phosphate buffer, pH 7.4 to a final concentration of 25 mg/mL. Place 20 µL of the enzyme solution on top of the cellulose acetate membrane and allow the water to evaporate (5-10 min).

Step 4. Cover the dried enzyme layer with a 4 cm2 membrane of polycarbonate membrane (0.05 µm pore size, 10 µm thick; Nucleopore) or general purpose dialysis tubing (MW cut off of 12,000-14,000). Fix the membrane with an O-ring (or silicone tubing).

Step 5. Trim off the excess membrane and place the probe in a 0.1 M phosphate buffer solution, pH 7.4 for 2 hr before use.

Useful life of the sensor will be several months if the probe is stored in room temperature in phosphate buffer. The steps involved are shown in Fig. 4.2.

4.1.3. Method 2: Crosslinking with glutaraldehyde

Generally higher loading of active enzyme can be obtained if the adsorbed enzyme is cross-linked with glutaraldehyde.

The method is the same as in Method 1 except one more step is added right after Step 3.

Step 3a. After the enzyme solution has dried, add 10 µL of a 1% solution of glutaraldehyde (Sigma Type I, supplied as a 25% solution which should be stored frozen and diluted in water immediately before use). Allow the glutaraldehyde solution to evaporate before proceeding to Step 4.

Variation: crosslinking with BSA

Mutual crosslinking of the enzyme with another protein such as bovine serum albumin (BSA; Sigma, Fraction V powder). This procedure can lead to higher enzyme activity and greater stability. Step 3 of Method 1 is modified:

Step 3. Prepare the glucose oxidase solution as in Step 3 of Method 1. Also, prepare 50 mg/mL BSA solution in the same phosphate buffer. Mix 10 L of each solutions and place the resulting 20 L on the cellulose acetate membrane. After 1-2 min. add 10 µL of 2.5% glutaraldehyde solution.The liquid layer should harden rapidly. After 1-2 hr, go to Step 4 (Method 1).

4.1.4. Method 3: Covalent attachment to membrane

Covalent attachment is most complicated but is useful in cases when the sensor is so small that the membrane must be fabricated directly on the sensing element. Covalent attachment gives more stable and reproducible enzyme activity.

Step 1. Dissolve 1.8 mg of cellulose acetate in a mixture of 20 mL of acetone and 3 mL of water.

Step 2. Cast 1 mL of this solution onto a clean dry glass plate using a spreader* (Touzart) and allow the solvent to evaporate for 1 min at room temperature.

*Note: The spreader has four channeled surfaces which yield films of 5, 10, 15, and 30 m thickness. A 15 m thickness is chosen.

Step 3. Remove the membrane by immersing the glass plate in distilled water and floating it off. The resulting membrane is cut in to smaller pieces and stored at room temperature in water.

Step 4. Suspend four membranes (each 2.5 cm square) in 100 mL of 0.1 M sodium periodate for 20 min at room temperature.

Step 5. Wash the membranes in distilled water for 5 min then immerse them in 10 mL of a 10 mg/mL solution of BSA in 0.1 M borate buffer pH 9 for 2 h.

Step 6. Remove 9 mL of the BSA solution and add 4 mg of sodium cyanoborohydride (Aldrich). Incubate at room temperature for 2 h.

Step 7. Wash the membranes in distilled water for 5 min and then store in phosphate-buffered saline at room temperature.

Step 8. Recrystallize p-benzoquinone (Merck) from petroleum ether and prepare a solution of 15 mg/mL in ethanol.

Step 9. Add 100 µL of the freshly prepared p-benzoguinone to 0.5 mL of a 20 mg/mL solution of glucose oxidase in 0.1 M phosphate buffer pH 7.4 in a tube covered by aluminum foil.

Step 10. Incubate the mixture for 30 min at 37_C and then remove the excess p-benzoquinone by gel filtration through a Saphadex C-25 column ( 1 x 10 cm) equilibrated with 0.15 M sodium chloride and operating at a flow rate of 20 mL/h. Collect the pink-brown band that elutes in the void volume (2-3 mL).

Step 11. Suspend the BSA-cellulose acetate membranes in 2-3 mL of the activated glucose oxidase solution after adjusting the pH of the latter to 8 -9 with 0.25 mL of 1 M sodium carbonate. Incubate at room temperature for 38 h.

Step 12. Remove the membranes, wash them by stirring in 0. 15 S M potassium chloride solution for 24 h and then store them in phosphate-buffered saline pH 7.4 containing 1.5 mM sodium azide.