Glucose Biosensor

Glucose Biosensor

Enzymes and Enzyme Technology
Glucose Biosensor


(Note that during this practical period you should make up the immobilised enzymes for next weeks practical exercise)
Aim

This practical is designed to give some insight into the preparation and properties of a biosensor.
Introduction

An amperometric sensor using glucose oxidase

Glucose oxidase is an enzyme produced and purified from Aspergillus niger which catalyses the oxidation of glucose.

Glucose + Oxygen --> delta- Gluconolactone + Hydrogen Peroxide

Glucose + Oxygen forward arrow d- Gluconolactone + Hydrogen Peroxide

This exercise demonstrates how glucose could be detected in a solution by use of a biosensor. Glucose analysis is very important in a number of diverse situations. It is of importance in the food industry for quality control purposes, in fermentation and, most importantly, as a clinical indicator of diabetes. It is for this latter purpose that so much current research has been directed at the perfection of a cheap, accurate and easy-to-use biosensor as the diabetic state is characterised by high levels of glucose in the blood (> 4.7 mM) and urine (> 11 mM). Glucose may be analyzed by a large number of different methods. However the method of choice usually involves an enzyme as such assays are very specific for glucose and, therefore, not easily interfered with by other sugars that may be present. In this practical exercise glucose oxidase will be used as the enzyme. It is very specific for glucose, oxidising it to gluconolactone in the presence of oxygen. The initial rate of this reaction is proportional to the concentration of glucose present, if sufficient oxygen is present in the solution, and may be determined from the rate at which oxygen is removed from the solution. The loss of oxygen is simply measured by use of an oxygen electrode. (This causes a very small proportion of the oxygen to be reduced to form water at its platinum cathode, producing a current which is proportional to the oxygen concentration). This exercise involves the assembly of a glucose biosensor with the glucose oxidase immobilised within a membrane at the surface of polarographic oxygen electrode. The biosensor is then used for the determination of unknown glucose solutions.

Before starting work, read through the Methods and Results sections.
Method

Preparation of the Immobilised Glucose Oxidase Membrane

glutaraldehyde crosslinks proteins
Safety note: The following makes use of glutaraldehyde, a bifunctional cross-linking reagent that reacts with lysine residues on the exterior of the proteins. Be careful not to get the glutaraldehyde on your skin as it may cause an irritation. If you spill your dilute solution, wash the area immediately in plenty of water.

1. Weigh about (~2 mg) 10 mg glucose oxidase (100 IU) and 60 mg bovine serum albumin into a test tube. Dissolve the mixture carefully in 1 ml of 20 mM phosphate buffer pH 7.0, by careful stirring (do not cause excessive foaming as this causes denaturation of the enzyme which reduces its activity).
2. Place a lens tissue on an exceptionally clean, flat and horizontal glass plate.
3. Carefully add 0.1 ml of 25% w/v glutaraldehyde to the protein solution. Mix. Carefully transfer the solution onto the lens tissue forming a single large damp area. Only add enough to dampen the complete tissue as excess is detrimental.
4. After allowing polymerising (this is not a drying process so do not heat or put in the sun) until set (about one hour), the membranes should be flooded with distilled water and carefully pealed away from the glass. If the membranes are still fragile at this stage then a further polymerisation period is indicated.
5. Place the membrane in a beaker containing 20 mM phosphate buffer pH 7.0 until needed.

Assembly of the Glucose Biosensor

Assemble the oxygen electrode with the addition of the glucose oxidase membrane to the top of the teflon membrane (i.e. facing the solution) as in the Appendix. Do not replace the capillary stopper, so allowing free access of oxygen from the air. Calibrate the biosensor as below using aliquots of 0.0~0.4 ml of 10 mg/ml glucose solution added to buffer to a final volume of about 2.5 ml:

1. Add some air saturated buffer (only) first to the oxygen electrode, as in the table below, and aerate thoroughly. Allow the oxygen electrode response to stabilize (at 100%) and start the chart recorder.
2. Add the required amount of standard glucose solution and note the changing response.

Sample
1 2 3 4 5 6 7
Air saturated buffer, ml 2.5 2.5 2.5 2.5 2.4 2.3 2.1
Add (10 mg/ml) glucose, ml 0.0 0.01 0.02 0.05 0.1 0.2 0.4

NB. Filling in the following table might help you calculate the glucose concentrations in each sample from the weight of glucose added and the total volume;

Sample


1


2


3


4


5


6


7

ml glucose added


0.0


0.01


0.02


0.05


0.1


0.2


0.4

therefore mg glucose added














1.0







total volume present; ml














2.5







final concentration; mg/ml














0.4







Now run your 'unknown' glucose sample by adding 0.2 ml of it to 2.3 ml of well-aerated buffer, as above. Note that the glucose actually determined is a dilution of the 'unknown'.

Plot a standard curve (which is usually a straight line) of the initial rate of oxygen depletion (change in oxygen concentration each minute, see below) against the final concentration of the glucose solutions (in mg/ml).

Rate of oxygen depletion

The glucose concentration of the (diluted) unknown solutions should be determined from the standard curve. Calculate the concentration of the original 'unknown' glucose solution by allowing for its dilution. Make sure that all your group has a copy of this data before you leave the laboratory.