CH324 Exp #7 Biosensor Glucose 20230327 (1)

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Feb 20, 2024

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Glucose Determination 7.1 Experiment #7 Oregon State University Version 20230327 Chemistry 324 GLUCOSE DETERMINATION USING AN AMPEROMETRIC BIOSENSOR  All solutions must be prepared and tested in the same day  Overview: This lab represents a departure from the standard instrumental analysis curriculum. In most instrumental analysis courses the experiments are limited to analytical techniques which can only be performed in the lab, usually with sophisticated and expensive analytical instrumentation. There are other classes of analytical devices, however, which are designed to be used outside of the lab. These are often used in the field for environmental applications, and increasingly for bioanalytical and biomedical applications. This experiment will cover the following areas; Electroanalytical devices – In this experiment we’ll be looking at an ana lytical device based on a sensor which is amperometric , i.e. the target compound is either oxidized or reduced and the analytical signal is measured as the amount of current produced by the sensor. Please read Chapter 17, page 357-8 in Harris for a more comprehensive description of this device. Biosensors – The term biosensor is most commonly used for an analytical device which is based on a signal generated using a biological molecule like an enzyme, antibody, DNA or RNA strand. Sometimes people will also refer to analytical devices which measure the concentration of a biological molecule as a biosensor. We’re going to study a diabetic glucose monitor as an example of a biosensor. The glucose monitor response is based on an enzyme which reacts specifically with glucose and very little else. The number of electrons produced (the current) can be directly related to the concentration of glucose. Consumer medical devices – There are a limited number of medical devices that the consumers can purchase which produce analytical information. The two most prominent examples are pregnancy tests, which produce a qualitative result (yes or no) and glucose monitors, which produce a quantitative result. An analytical device which can produce an accurate and precise result, when used by a consumer of unknown educational background and with little or no training, is difficult to design. We’ll be looking at the precision, accuracy and limitations of one of these monitors. Background: Diabetes affects millions of Americans, and costs the healthcare system billions of dollars, every year. Chronically high or low glucose concentrations can both have long term health consequences. That makes the monitoring of blood glucose concentrations in diabetics a critical health issue. Insulin dependent diabetics will take shots of insulin several times a day based on the quantitative analytical result produced by one of these glucose meters. A single bad analytical result that causes a diabetic to take too much insulin will result in hypoglycemia , which can be acutely fatal. A series of bad analytical results that causes a diabetic to take too little insulin over a longer period of time can result in chronic hyperglycemia , which can result in a diabetic coma and death. Figure 1 (an adaptation of the Clarke Error Grid) shows a hypothetical glucose sensor response, and the allowable error based on the medical action that would be taken as a result of the analytical answer. You will notice that the allowable error changes dramatically based on where you are in the calibration curve. If the patient is hypoglycemic the difference between 30 and 50 mg/dL is rather unimportant since the medical action – immediately giving the patient glucose – is the same regardless of what the true value is. If the patient is hyperglycemic, the difference between 350 and 400 mg/dL is unimportant since the patient will immediately be hospitalize, and the medical staff will give them insulin in small doses to lower the patient’s blood glucose level gradually, rechecking the concentration many times while they’re administering insulin. It’s important to lower concentrations slowly because a sudden drop in glucose can elevate potassium concentrations, resulting in a heart attack.

Glucose Determination 7.2 Experiment #7 Oregon State University Version 20230327 Chemistry 324 Glucose Biosensor Response y = 0.9964x + 0.9674 R 2 = 0.9963 0 50 100 150 200 250 300 350 400 450 0 50 100 150 200 250 300 350 400 450 Glucose concentration (mg/dL) Signal (nA) Figure 1: Glucose Biosensor Response, With Error Limits Derived From the Clarke Error Grid How good is an analytical instrument that costs under $100, and requires no training or lab skills to operate? That’s the question you’ll answer in this lab. The technology: A glucose monitoring system has two components;  A single use test strip which contains the glucose specific enzyme, an electron mediator and electrodes.  A meter which measures sensor current and, after calculations using a stored calibration curve, displays the glucose concentration. Figure 2 shows a schematic of how the chemistry in the strip works. Electrochemical processes, and many biological processes, are oxidation/reduction reactions. In an oxidation/reduction reaction, the molecule being oxidized loses electrons, while the molecule being reduced gains electrons. For every oxidation there must be a corresponding reduction. The strip chemistry starts when the sample containing glucose encounters the glucose enzyme, in this case glucose oxidase (GOD). Glucose is oxidized to gluconolactone while the enzyme, GOD, is reduced. A reagent already in the strip, the mediator, re-oxidizes the GOD allowing it to oxidize another molecule of glucose. When the mediator oxidizes the GOD, the mediator is reduced. Finally, at the electrode surface the mediator is re-oxidized, and the meter counts how many electrons are produced. The number of electrons is proportional to the glucose concentration in the blood sample. Allowable Error

Glucose Determination 7.3 Experiment #7 Oregon State University Version 20230327 Chemistry 324 Figure 2: Schematic of the Chemistry in an Electrochemical Glucose Test Strip Each production lot of test strips is calibrated during the manufacturing process to provide an accurate result for a whole blood sample, which contains very specific concentrations of salts, lipids (fats), proteins, and red blood cells. Since we’l l be using aqueous standards which are missing most of these components, the meter reading will not accurately report the true glucose concentration in your solutions. You’ll be forced to construct a calibration curve to convert the meter readings (for whole blood) into a corrected value for an aqueous solution. Your calibration plot will look something like the data in Figure 3; Glucose Gluconolactone (Ox) Glucose Dehydrogenase (Ox) Glucose Dehydrogenase (Red) Ferrocyanide (Red) Ferricyanide (Ox) Anode Surface e - e - e - e - Counter Micro-processor Display Target Molecule Enzyme Mediator

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Glucose Determination 7.4 Experiment #7 Oregon State University Version 20230327 Chemistry 324 Glucose Meter Calibration y = 1.2454x + 1.2093 R 2 = 0.9963 0 50 100 150 200 250 300 350 400 450 500 0 50 100 150 200 250 300 350 400 450 Glucose Standards Concentration (mg/dL) Glucose Meter Reading (mg/dL) Figure 3: Calibration Plot of Glucose Meter Response versus Standards Concentration Experimental Procedure: This experiment has a surprisingly low first time pass rate. Most of the problems seem to be connected to mis-use of pipettes. Please check with a TA and make sure that you clearly understand the proper procedure for using pipettes. Bring a 50 mL beaker and a 250 mL beaker to the TAs. You’ll be given a 2.00 g/dL glucose standard solution and a special pH/ionic strength diluent for your solutions. The volume “ deciliter ” is not commonly used by chemists, but is used for many measurements in clinical chemistry. If you ’ re unfamiliar with the term, please look it up. The normal concentration range for fasting non-diabetics is 80- 120 mg/dL. The standard solution concentrations you’ll be making are representative of the concentration range found in diabetics whose blood glucose level is not well controlled. From the standard solution you’ll make up 50.00 mL of a series of standard solutions with the following concentrations; 80 mg/dL 120 mg/dL 160 mg/dL 200 mg/dL To make these dilutions you should use volumetric pipettes. When you make your dilutions, use the special ionic strength/pH buffer solution provided for the experiment, not DI water . You’ll need about 250 mL. Bacteria grow very quickly in these solutions so we store them in the refrigerator. Since enzymes reactions are extremely temperature sensitive, make sure that all of these solutions are at room temperature before you run your analysis . If you’re uncertain on how to obtain these concentrations from the standard solution remember the standard dilution equation: conc 1 x vol 1 = conc 2 x vol 2 High Conc Pipette Vol Low Conc Flask Vol

Glucose Determination 7.5 Experiment #7 Oregon State University Version 20230327 Chemistry 324 When calculating a dilution, once you pick the size volumetric flask to use for the new solution you always know three out of four of the values. If you’re still uncertain about the dilutions check with a TA a fter you’ve made your calculations. Once you’ve made your standards , return to the TA with your notebook and a weighing bottle to obtain your unknown, the glucose test strips and the diabetic glucometer. Each test strip can only be used once, so you’ll ne ed 7 test strips. Have the TA record in your notebook the unknown number AND the meter number. Keep the top on your weighing bottle to prevent evaporation. Make sure that the test strip lot numbers ALL match each other. Insert a test strip into the glucometer (check with a TA if you need assistance). Place a small volume of each standard in a small clean, watch glass . You just need to touch the strip to the surface of your solution (vertically) and the strip will obtain the correct volume by capillary action. Immersing the strip in the solution will produce an inaccurate result. Each of your standards will be tested once . In about 5 seconds the meter will display a glucose concentration value in mg/dL (you will hear a “ beep ” ). Record the value, replace the test strip and repeat for the next solution. Of course the meter readings of your standards WILL NOT match your concentrations since you’re not using whole blood as a sample. After you’ve tested all of your standards, place a small volume of your unknown on a clean watch glass and test it in triplicate. Standards and unknowns must be run at the same time (+/- an hour). You cannot use calibration data from a previous lab period. Plot your calibration curve and find the Linear Least Squares (LLS) best fit line, being sure to display the LLS equation. The current slope and intercept may vary with the manufacturing lot of the sensor strips. Check with the TAs for the approximate slope and intercept for the current lot . The incorrect slope or intercept, or a non-linear curve, are signs that the pipettes were used incorrectly. Print out your calibration curve and include it in your notebook. Use that LLS equation to find the concentration of glucose in the unknown solution. Report: All experimental data, calculations, graphs and calibration curves must be in your notebook (extra pages stapled) BEFORE you submit an answer to the computer system. Failure to do so will result in the loss of 10 points from your course score. Submitting answers not supported by experimental data recorded in your notebook will result in IMMEDIATE dismissal from the class with a grade of F. Be prepared to answer the following questions before you log on to the lab computer; 1. What is the % relative standard deviation (StDev/Average Reading * 100%) of the unknown solution? Clinical chemists often refer to this value as the coefficient of variation (%CV). 2. What is the concentration of glucose in the unknown solution in mg/dL? 3. What is the concentration of the unknown solution in moles/L (the formula weight of glucose is 180 g/mole)? 4. The newest glucose test strips are designed to operate on extremely small volumes of blood. If the strip requires 0.6 uL of sample, how many moles of glucose were in the sample analyzed by the strip?

Glucose Determination 7.6 Experiment #7 Oregon State University Version 20230327 Chemistry 324 Waste Disposal None of the waste from this experiment is hazardous. Solutions may be poured directly down the sink and test strips may be thrown in the garbage. Acknowledgement OSU wishes to acknowledge the help provided by Dr. Tim Henning of Abbott Diabetes Care in the design of this lab. Standard Solution: 20.00 g/L glucose, 0.15 M NaCl, 0.01 M NaH 2 PO 4 , 0.01 M Na 2 HPO 4 Diluent: 0.15 M NaCl, 0.01 M NaH 2 PO 4 , 0.01 M Na 2 HPO 4

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