Enzyme Immobilization Protocol

Enzyme Immobilization and Membrane Application Protocol

This protocol details the procedures for immobilizing enzymes on the dual-electrode sensor and applying protective membranes. This is a critical step that determines the sensor's selectivity, sensitivity, and stability.

1. Safety Precautions

Before beginning the enzyme immobilization process, ensure the following safety measures are in place:

• Wear appropriate personal protective equipment (PPE) including laboratory coat, nitrile gloves, and safety goggles.
• Handle glutaraldehyde in a fume hood with appropriate ventilation due to its irritant properties and potential toxicity.
• Handle organic solvents (e.g., tetrahydrofuran) in a fume hood.
• Enzymes may cause allergic reactions in sensitive individuals; handle with care.
• Review Material Safety Data Sheets (MSDS) for all chemicals used in this protocol.

2. Materials and Equipment

2.1. Materials

• Nanomaterial-modified dual-electrode sensors (prepared according to the Fabrication Protocol)
• Glutaminase (from E. coli, lyophilized powder, ≥40 units/mg protein)
• Glutamate oxidase (from Streptomyces sp., lyophilized powder, ≥5 units/mg solid)
• Bovine serum albumin (BSA, lyophilized powder, ≥96% purity)
• Glutaraldehyde (25% solution in water)
• Nafion (5% w/w solution in lower aliphatic alcohols/water mixture)
• Polyurethane (medical-grade, aliphatic polyether urethane)
• Tetrahydrofuran (THF, anhydrous, ≥99.9% purity)
• Phosphate buffered saline (0.1 M PBS, pH 7.4)
• Deionized water (resistivity ≥18.2 MΩ·cm)
• Triton X-100 (non-ionic surfactant, for hydrophobic electrode wetting)

2.2. Equipment

• Analytical balance (readability 0.1 mg)
• Micropipettes and tips (various volumes: 0.5-10 μL, 10-100 μL, 100-1000 μL)
• pH meter
• Vortex mixer
• Refrigerator (2-8°C) and freezer (-20°C)
• Laboratory oven or incubator with temperature control
• Fume hood
• Fine-tip tweezers
• Timer
• Microcentrifuge tubes (1.5 mL)
• Ice bath
• Humidity chamber (can be improvised using a sealed container with wet paper towels)

3. Preparation of Solutions

3.1. Enzyme Solutions

Note: Prepare enzyme solutions fresh on the day of use. Keep enzymes and solutions on ice during preparation to maintain activity.

3.1.1. Glutaminase Solution (10 U/mL)

1. Calculate the amount of glutaminase powder needed based on its specific activity (units/mg).
2. Weigh the required amount of glutaminase using an analytical balance.
3. Dissolve in cold (4°C) 0.1 M PBS (pH 7.4) to achieve a concentration of 10 U/mL.
4. Add 10 μL of 0.1% Triton X-100 per 1 mL of solution (final concentration: 0.001%).
5. Mix gently by inversion; avoid vigorous shaking to prevent denaturation.
6. Keep the solution on ice until use.

3.1.2. Glutamate Oxidase Solution (5 U/mL)

1. Calculate the amount of glutamate oxidase powder needed based on its specific activity (units/mg).
2. Weigh the required amount of glutamate oxidase using an analytical balance.
3. Dissolve in cold (4°C) 0.1 M PBS (pH 7.4) to achieve a concentration of 5 U/mL.
4. Add 10 μL of 0.1% Triton X-100 per 1 mL of solution (final concentration: 0.001%).
5. Mix gently by inversion; avoid vigorous shaking to prevent denaturation.
6. Keep the solution on ice until use.

3.2. BSA Solution (5% w/v)

1. Weigh 50 mg of BSA using an analytical balance.
2. Dissolve in 1 mL of cold (4°C) 0.1 M PBS (pH 7.4).
3. Add 10 μL of 0.1% Triton X-100 per 1 mL of solution (final concentration: 0.001%).
4. Mix gently until completely dissolved.
5. Keep the solution on ice until use.

3.3. Glutaraldehyde Solution (2.5% v/v)

1. In a fume hood, add 100 μL of 25% glutaraldehyde solution to 900 μL of cold 0.1 M PBS (pH 7.4) in a microcentrifuge tube.
2. Add 9 μL of 0.1% Triton X-100 (final concentration: 0.001%).
3. Mix gently by inversion.
4. Prepare fresh and use within 1 hour.

3.4. Polyurethane Solution (2% w/v)

1. In a fume hood, weigh 20 mg of polyurethane using an analytical balance.
2. Add to a glass vial containing 1 mL of THF.
3. Cap the vial tightly and allow the polyurethane to dissolve overnight at room temperature.
4. The solution should be clear; if not, allow more time for dissolution.
5. Store at room temperature in a tightly sealed container (stable for up to 1 week).

4. Enzyme Immobilization Procedure

4.1. Preparation of Enzyme-BSA Mixtures

4.1.1. Mixture for Electrode A (Glutaminase + Glutamate Oxidase)

1. In a microcentrifuge tube on ice, add:
   • 20 μL of glutaminase solution (10 U/mL)
   • 20 μL of glutamate oxidase solution (5 U/mL)
   • 10 μL of BSA solution (5% w/v)
2. Mix gently by pipetting up and down several times.
3. Keep on ice until use.

4.1.2. Mixture for Electrode B (Glutamate Oxidase Only)

1. In a microcentrifuge tube on ice, add:
   • 20 μL of glutamate oxidase solution (5 U/mL)
   • 20 μL of 0.1 M PBS (pH 7.4)
   • 10 μL of BSA solution (5% w/v)
2. Mix gently by pipetting up and down several times.
3. Keep on ice until use.

4.2. Enzyme Immobilization by Cross-linking

1. Label the dual-electrode sensor to identify Electrode A (glutaminase + glutamate oxidase) and Electrode B (glutamate oxidase only).
2. Place the sensor on a flat, level surface in a humidity chamber.
3. Using a micropipette, carefully deposit 3 μL of the Electrode A enzyme-BSA mixture onto the surface of Electrode A.
4. Similarly, deposit 3 μL of the Electrode B enzyme-BSA mixture onto the surface of Electrode B.
5. Allow the enzyme-BSA mixtures to spread evenly across the electrode surfaces.
6. Close the humidity chamber and allow the mixtures to partially dry at room temperature for 5 minutes.
7. In a fume hood, add 1 μL of 2.5% glutaraldehyde solution to each electrode surface.
8. Close the humidity chamber and incubate at room temperature for 1 hour to allow cross-linking to occur.

5. Membrane Application

5.1. Nafion Membrane (Inner Selective Layer)

1. After the cross-linking incubation, rinse the electrodes very gently with a few drops of cold 0.1 M PBS to remove any unbound material.
2. Allow the electrodes to dry at room temperature for 10 minutes.
3. Using a micropipette, carefully deposit 2 μL of 5% Nafion solution onto each electrode surface.
4. Allow to dry at room temperature for 30 minutes, or in an oven at 40°C for 15 minutes.
5. The Nafion membrane serves as a selective barrier that reduces interference from negatively charged species.

5.2. Polyurethane Membrane (Outer Protective Layer)

1. In a fume hood, using a micropipette, carefully deposit 2 μL of 2% polyurethane solution onto each electrode surface.
2. Allow to dry at room temperature for 1 hour, or in an oven at 30°C for 30 minutes.
3. Repeat steps 1-2 to apply a second layer of polyurethane.
4. Allow the final layer to dry completely at room temperature for at least 2 hours.
5. The polyurethane membrane serves as a protective barrier that limits diffusion and extends sensor lifetime.

6. Conditioning and Storage

1. After the membranes have dried completely, soak the sensors in 0.1 M PBS (pH 7.4) for 2 hours at room temperature.
2. This conditioning step helps to stabilize the enzyme layers and membranes.
3. After conditioning, store the sensors at 4°C in 0.1 M PBS (pH 7.4) when not in use.
4. For long-term storage (>1 week), store the sensors dry at 4°C in a sealed container with desiccant.

7. Quality Control

The following quality control checks should be performed on a representative sample of sensors from each batch:

7.1. Visual Inspection

• The enzyme and membrane layers should appear uniform without cracks or delamination.
• There should be no visible contamination or debris on the sensor surface.

7.2. Enzyme Activity Check

For a representative sensor from each batch:

1. Prepare a solution of 1 mM hydrogen peroxide in 0.1 M PBS (pH 7.4).
2. Apply a drop of this solution to the sensor and observe for bubble formation (oxygen release) on the electrode surface.
3. Bubble formation indicates that the glutamate oxidase is active.
4. Note: This is a qualitative check and does not replace full functional testing.

8. Troubleshooting

Problem	Possible Cause	Solution
Poor enzyme immobilization (low activity)	Enzyme denaturation during preparation; insufficient cross-linking	Keep enzymes cold during preparation; optimize glutaraldehyde concentration and cross-linking time
Membrane cracking or peeling	Too rapid drying; excessive thickness	Control drying conditions (slower drying at lower temperature); optimize membrane solution volume
Uneven membrane coverage	Uneven application; surface contamination	Ensure level surface during application; clean electrode surface thoroughly before enzyme application
Cross-contamination between electrodes	Solution spreading during application	Apply smaller volumes; ensure adequate spacing between electrodes; use precision micropipettes
Rapid loss of enzyme activity	Insufficient cross-linking; improper storage	Optimize cross-linking conditions; store sensors properly (4°C, hydrated or with desiccant)

9. Next Steps

After successful enzyme immobilization and membrane application, proceed to the "Sensor Assembly and Quality Control Protocol" for the next phase of sensor development.

Note: Document all observations, measurements, and deviations from the protocol in a laboratory notebook. Record the specific enzyme activities, lot numbers, and expiration dates for traceability and troubleshooting purposes.