Glucometry

BIOE 415 Lab 7

Introduction

Glucose concentration in blood is measured several times daily by the millions of diabetics in the world. Most use inexpensive glucose meters and disposable test strips. A small amount of blood is drawn to an electrode on the test strip where a series of chemical reactions takes place. The electrons produced by these reactions are measured as either current or total charge. The measured value is proportional to the concentration of glucose over a wide range. To nd the concentration the meter compares the measured value to that from a calibration standard. The test strip is impregnated with glucose oxidase (GOX), which is reduced in the presence of glucose. The reduced GOX then reduces a mediator (M) and becomes oxidized. Finally, the mediator becomes oxidized (due to an applied voltage) and produces electrons. It is these electrons that are measured. glucose + GOXox GODred + 2Mox 2Mred gluconolactone + GOXred + H2 O2 GODox + 2Mred + 2H + 2Mox + 2e

A glucometer uses three operational ampliers arranged as a potentiostat (see Figure 1). The test strip has three electrodes. The working electrode is the electrode at which electrons are produced. It is connected to a current-to-voltage amplifer (with optional capacitor to lter high frequencies). The other two electrodes are the reference electrode and the counter electrode. The reference electrode should be held at a constant voltage (of around -400 mV compared to the working electrode in this case) to push the reactions outlined above. This induces a current through the resistive electrolyte. The working electrode voltage is reduced by the voltage now across the electrolyte. The reference electrode in Figure 1 is connected to the input of an op amp so very little current can ow from the reference to the working electrode. Some current must ow, however, so that the reactions above can take place. This current ows between the counter and working electrodes. An operational amplier ensures that the counter electrode can supply enough current. Also, if the reference voltage should change, the counter electrode op amp will quickly react to the change by supplying more or less current and bringing the reference voltage back to its initial value. The electrodes on One Touch Ultra glucose test strips (Lifescan Inc.) are arranged as shown in Figure 2. A simplied glucometer is shown in Figure 3. This device uses only two of the electrodes so the reference electrode may not be maintained at exactly 400mV . It is dicult to make connections to all three electrodes in lab, so we will use this conguration that uses only two. In this lab students will build the simplied potentiostat and test two or three glucose solutions of known concentrations. A calibration curve will be made with the data. Glucose test strips degrade over time, especially if exposed to air. Additionally they are fairly expensive ($1 each). Please use only two test strips and keep their container closed as much as possible.

Pre-lab assignment
1. Read the lab procedure. 1

R=82k + 10V Rref 3.3k vref LM358 + ref. working i Rf=1M LF356 + vout counter LM358 +

Figure 1: A potentiostat for use in a glucometer.

(a) One Touch Ultra test strip electrode conguration.

(b) Be sure to ll the test strip from the edge and to completely ll the window.

Figure 2: One Touch Ultra test strip information.

working

reference

R2 R1 + 10 V LM358 -400mV +

100k LF356 + vout

Figure 3: A simplied potentiostat for use in a glucometer.

2. For the circuit in Figure 3 do the following: choose resistor values (R1 , R2 ) so that vref = 400mV choose C so the output op amp attenuates signals higher than 50Hz choose values for the low pass lter at the output to once again attenuate signals higher than 50Hz nd vout given i, the current into the working electrode 3. Read the specication sheets for the LM358 and LF356. What is the input bias current for each device? Given that what would you consider to be the smallest current i that each could reliably measure?

Lab Procedure
1. Build the potentiostat of Figure 3. 2. As a test, connect a 10k resistor between the reference and working electrodes. Apply a square wave that oscillates from 0 10V at 20Hz and measure the output. What is the output? 3. Before testing your device with test strips set up your oscilloscope to display several seconds and a range of 1 1.5V . Also, be ready to hit the STOP button so that you can save the output voltage curve. Finally, remove the square wave excitation and replace it with 10V from the DC power supply. 4. Each group will receive only two test strips, one for low glucose concentration (2.8mmol/L) and one for high glucose concentration (16.7mmol/L). The test strip manufacturer claims that the strips can measure 1.1 33.3mmol/L. When you are ready to test, get a test strip and a bottle with glucose solution (either low or high) from your TA. Attach the test strip to the miniature test clips and put a paper towel under it. Use a small pipet to bring a small drop of glucose solution to the top edge of the test strip. The sample will enter the test strip via capillary action. Be sure to completely ll the window on the test strip. 5. As soon as the glucose hits the electrodes the voltage output should start to increase. When it peaks and starts to go back down, hit STOP on the oscilloscope and save the voltage curve to the computer. 6. Choose a time after the curve begins on both traces and use the voltage at that time as the output. Construct a plot comparing glucose concentration to output voltage at the time you chose.

Clean-up

Please be careful with the glucose as it is messy. If any is spilled clean it up with a paper towel. Through away any used test strips.

Post-lab
1. Answer any questions that appear in the lab procedure. 2. Please read Electrochemical glucose biosensors by Joseph Wang in Chemical Review vol. 108(2), February 2008 and answer the following questions. (a) What dierentiates the three generations of glucose sensors? (b) What is a closed-loop or sense and act system? (c) What are the requirements for in vivo glucose monitoring?