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Computer Data Acquisition for Biochemistry Practice Works
Methods and Examples
How to make data acquisition from a Clark electrode using a Metex multimeter, piloted by a PC with the Datalyse software


This page describes an easy and cheap method to make numerical data acquisition from a palarographic oxymeter signal. Leland C. Clark described in 1962 a polarographic device the " Clark electrode ", allowing to measure the glucose in blood, by following the decrease of dissolved O2, due to the glucose oxydase enzyme activity. L.C. Clark is generally considered as the inventor of the first biosensor.

The principle of the method:

Briefly, the probe consists in an anode (Pt) and a cathode (Ag) kept in contact with a thin bridge of paper, soaked with a saturated solution of KCl. This electrolytic bridge and the anode are enclosed below a membrane (Teflon or polypropylene) permeable to O2 but not to irons or organic compounds. This membrane is polarized by a control device (about 0.6 V). O2 is soluble in water, and by diffusion through the membrane, can react with the Ag of the cathode. The current of this oxydo-reduction (at the constant polarizing voltage of 0.6 V) is directly proportional to the partial pressure of oxygen diffusing to the reactive surface of the electrode. The controle module converts this current into a tension which allows to measure the variations of O2 concentrations. More details about the physico-chemical principle are avaible on the "Experimental Biosciences" web site.

The device with the acquisition montage: the Metex M-3850D RS-232 port is connected to a USB port computer using a RS232 to USB adaptor (see How to use Datalyse on a USB computer, How to use Datalyse on a USB Apple Macintosh notebook, for computer compatibilities, and Metex family description on Datalyse website). The detailed mounting preocedure of the probe is descibed step by step in the page "Clark probe - pratical note".

A set of quick experiments showing the quality of result you could expect from this device:

Here is an example of the acquisition of an Glucose Oxydase (GO) activity: The kinetic was followed during 10 minuts with an acquisition every 5 seconds. Note the initial linear speed of the catalyse, following by a nonlinear step due to the decrease of the O2 concentration. The concentration of the dissolved oxygen in water, is a limit of the method. (contents of the cell; 2 ml of 0.5 M of beta-D-glucose, with about 0.05 UI of GO, pH 5 in 50 mM phosphate buffer, 25°C). In these conditions, the glucose concentration is in large excess comparing to the Michaelis-Menten constant.

The next picture shows three kinetics, with variable amounts of GO enzyme (same conditions than above). Note that the time of linear reaction decreases strongly with the amount of enzyme.

 

This representation of the precedent experiment, shows the good linearity obtained between the measured activity and the amount of GO: a two fold increase of GO amount, results in about a two fold increase of measured activity. The slope obtained gives a specific activity of about 270 UI/mg at 25°C which is in accordance with this announced by the supplier (340 U/mg at 35°C).

The next set of experiments shows the determination of the Michaelis-Menten constant, by using decreasing concentrations of glucose (from 0.5 M to 8 mM, with 50 mUI of GO, in the same conditions described above). The device allows to measure some activities in the order of 10 mV/min.

This representation of the mesures shown above, gives a good fitting with a hyperbolic model:

The double inverse representation of Lineweaver-Burck, shows a good linarity and allows to deduce a maximum speed of about 53 nmol/min and a Michaelis-Menten constant of 57 mM, which is in the range of the known values from 33 to 110 mM (source Sigma chemicals)

Products:

Glucose Oxydase from aspegillus niger from Sigma, G.9010.

beta-D-Glucose from Prolabo, 24379.294.

Special thanks to Alessandra Albano for her participation to the English correction of this page.

Gilles Carpentier's Web Site: "Computer Data Acquisition for Biochemistry Practice Works: Methods and Examples" 

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