Utilization of the CBL^Ô System
For
Glucose & Protein Analysis of Blood Serum

Craig T. Gabler
Chemistry/Physics Teacher, Centralia High School
(cgabler1@esd113.wednet.edu)
in cooperation with
Dr. Benard Van Wie, Mentor, WSU, and Nancy Davis, Middle School Teacher, Connell, WA

Table of Contents

1. Introduction…………………………………………………. 1

    

2. Teaching Strategies
   1. Task Analysis .………………………………………..2
   2. Timeline ………………………………………………3
   3. Teaching Suggestions ………………………………..3
   4. Lab Hints …………………………………………….5

    

3. Student Version of Laboratory Investigation ………………..7

    

4. Sample Lab Report ………………………………………….13

    

5. Appendices

1. Blood Chemistry Background Information ……………..14
2. Sources of Materials ……………………………………15
3. TI-82 Graphic Analysis Instructions ……………………16
4. Washington State Essential Academic Learnings ………17
5. Transparency Masters…………………………………..18

I. Introduction

Most pre-college mathematics and science teachers are very familiar with the idea that science represents a body of knowledge, and is a method for expanding that body of knowledge. However, they are likely to be less familiar with the field of engineering and the nature of the work done by engineers. It is this situation that provided the direction for the WSU/NSF Summer Teacher’s Institute, held on the campus of Washington State University and hosted by the Department of Chemical Engineering. The goal of the Institute was to provide experiences to math and science teachers showing that engineering involves putting matter and energy to use for society, by the practical application of scientific knowledge. Using practical applications, the engineer is typically asked to create a device or system that will do something, better, faster, and/or cheaper. In addition to learning about engineering, the Institute participants were asked to prepare a classroom module that introduces or integrates engineering principles into the existing pre-college curriuculum.

This particular module was created in cooperation with the research being done by Dr. Bernard Van Wie and his team of graduate students. The goal of their research is to develop a multifunctional blood analyzer which utilizes a flow injection analysis approach. The blood analyzer is being designed to perform the eight most common clinical tests rapidly, automatically, less expensively, and in a more compact form than is currently available. To create this blood analyzer the researchers have to first understand the fluid mechanics of the system and determine the reaction kinetics for the system. Because the development of the automated blood analyzer represents the integration of basic scientific knowledge, engineering principles, and applications of technology it is a natural for carry-over to the pre-college classroom.

In the module that follows, the focus is on providing students with a hands-on laboratory investigation of the concentration of glucose or protein in a simulated blood serum. The same reagents as Dr. Van Wie’s group uses will be utilized, but rather than employing the same elaborate spectrophotometric system, the students will use a Texas Instruments CBL in conjunction with a simple colorimeter. While the protocols for preparing the solutions were created during the Institute, the overall procedure is an adaptation of the colorimeter labs found in the Vernier Software book, Chemistry with CBL. In the investigation the students will prepare a calibration curve, and then from that determine the concentration of their simulated blood serum sample. This application of basic chemistry and mathematics principles is an excellent opportunity for students to learn inference building. As an additional outcome of this investigation students will have the knowledge and skills necessary to conduct an independent research project of their own choosing.

The goal of this module, through the testing of simulated blood serum for glucose and protein, is to provide students with the knowledge and skills associated with an authentic chemical analysis. In addition, students will have the opportunity to acquire the background necessary to carry out an independent research project of their own design.

The student should be able to:

1. Understand the importance of blood chemistries (1,4).
2. Apply basic chemical reaction principles to biological systems (4,5).
3. Determine serum glucose levels using the CBL system (1,2).
4. Determine serum protein levels using the CBL system (1,2).
5. Apply graphing skills to the interpretation of data (4).
6. Compare clinical glucose levels to normal levels and predict hypoglycemia or hyperglycemia (1,4,5).
7. Compare clinical protein levels to normal levels and predict hypoalbuminemia or hyperalbuminemia (1,4,5).
8. Apply Beer’s Law to CBL/colorimetric data (1,4,5).

The numbers in parentheses following each objective refer to a Washington State Essential Academic Learning Requirement (EALR). See Appendix D for a list of science EALR’s.

In order for the students to conduct these investigations successfully it is necessary that they have previously acquired the knowledge and/or skills to:

1. Prepare a graph from raw data.
2. Utilize the CBL system for data collection.
3. Determine the slope and intercept of a straight line on a graph.
4. Interpret basic chemical reactions.
5. Express concentration using various methods.

In preparation to teach this module, or any science concept, it is helpful to look at an analysis of the task we are asking the student to perform. In light of the aforementioned student learning objectives, and the stated prerequisites, a task analysis was performed and formatted into a hierarchy chart (see next page). A careful review of this will help you better prepare yourself and the students to carry out this module.

Day 1: Introduction to Blood Chemistry - glucose & protein levels and symptoms
Day 2: Background on colors, absorbance/transmittance, and Beer’s Law
Day 3: Pre-lab Day - describe equipment set up, chemical reactions, and safety
Day 4: Lab Day
Day 5: Analysis of Data
Day 6: Post Lab Discussion
Day 7: Lab report due - visit by doctor

C. Teaching Suggestions

1. Appendix A contains background information concerning blood chemistry and an excellent, but technical reference. Another possibility for introducing this unit is to invite a nurse or doctor in to discuss the role of blood and its components.

    

2. To introduce the concept of what is happening to light as it passes through a colored substance one could use various colored transparencies, colored cellophane wrap, or even colored Saran wrap. Make certain the students understand the connection between the color we see as being the transmitted color, and all the other colors of the rainbow as being the absorbed colors.

    

3. To help develop an understanding of the relationship between how much colored substance is present (concentration) and how much light is absorbed/transmitted (Beer’s Law) one could progessively add more and more food coloring to a beaker placed on the overhead projector.

    

4. The transparency masters found in Appendix E can be used to help students see the relationship between the color of light being absorbed and the actual color of the solution they are observing.

    

5. This module provides the students with a nice set of laboratory skills that could allow them to pursue some independent research projects. Some ideas are: the comparison of glucose in various beverages, the comparison of protein content of beverages like milk or some of the diet drinks, or the use of glucose reagent for a kinetic study of the break down of starches by the salivary enzyme amylase.

6. Measurement of the glucose in Pepsi found that a typical Pepsi contained 6.44 g/dL glucose, or 55.8% of the sugar present. This is consistent with food labeling, because the Pepsi contains ‘high fructose’ sugar, which is generally taken to be 58% glucose and 42% fructose. In the case of skim milk, previous research found it to contain 23 g/L protein.

D. Lab Hints

1. This module is designed to have students work in pairs in the lab. If you are going to have students perform both the glucose assay and protein assay it is recommend that you assign half of the groups to do a protein assay and the other half to do the glucose assay. This will reduce the volume of glucose reagent required, and thereby make the lab experience much more cost effective.

    

   If you have two CBL/colorimeter setups it is recommended that one setup be used for the protein assay and the other for the glucose assay. A cuvette containing the blank must be provided at each colorimeter. In the case of the glucose assay the blank can be made from 10 drops of water and 2.5 mL of glucose reagent. The protein assay blank should be 8 drops of buffer added to 3.0 mL of Bradford reagent.

   The simulated serum samples can be distributed in small test tubes or labeled pipets. The students should be careful to record the identification letter of their serum sample in their lab report. Be sure to notify students that the serum samples are a diluted form.

2. Preparing solutions (sufficient quantities for 50 pairs of students) -

glucose - 60 mg/dL - Dissolve 0.3 g of glucose in 500 mL of distilled water. Students then dilute this to prepare their glucose solutions. The following example illustrates how the concentrations are computed (this calculation is worth pointing out to students):

glucose reagent - Make up per instructions on bottle, keep refrigerated.

buffer - Dissolve 7.65 g NaCl, 1.26 g Na₂HPO₄, & 0.1 g NaH₂PO₄ in 1.0 L.

albumin - 0.1 g/dL - Dissolve 0.05 g Bovine Serum Albumin (BSA) in 0.5 L of buffer. Student concentrations computed the same as for glucose.

Bradford reagent - 0.025 g of Coomassie Blue. Add 125 mL of ethanol. Dissolve well then add 25 mL of concentrated phosphoric acid. Filter. Keep refrigerated. Filter before each use.

Serum Stock Solution - dissolve 0.10 g BSA and 0.15 g glucose (dextrose) in one liter of buffer. Keep refrigerated.

Serums - The following mixtures represent a 1:50 dilution for glucose and a 1:1000 dilution for protein, from what the concentrations would be in straight serum samples. Students must account for this in their calculations for the actual glucose or protein concentration.

 

Serum Sample	A	B	C	D
mL serum stock solution	90	70	50	30
mL buffer	10	30	50	70
[glucose] mg/dL	13.5	10.5	7.5	4.5
[albumin] mg/dL	9	7	5	3

3. Safety

Standard laboratory precautions need to be discussed. All solutions can be disposed of by flushing them down the drain with plenty of water. Emphasize that the serum the students are using is a laboratory prepared serum, not real human or animal serum.

4. Adaptations

1. This investigation could be carried out by each group if sufficient CBL/colorimeter setups are available. This could also allow each group to perform both the glucose assay and the protein assay.

2. This investigation can still be carried out if only 1 CBL/colorimeter setup is available by either spreading the lab over two days, or limiting it to one or the other of the assays.

3. If the CBL/colorimeter is not available, but a spectrophotometer such as a Spec20 is, the procedure is easily adapted.

 

When you visit your doctor for a check up, blood may be drawn as part of the examination. Have you ever wondered what they did with that blood, besides counting the number of red blood cells? Depending on what the doctor needs to know, he or she may request that the blood be tested for a number of substances, such as glucose, albumin (a protein), or cholesterol. Have you ever wondered how they measure the concentrations of these different substances? Well, wonder no more.

In this lab you are going to utilize one of the most modern methods for determining blood glucose and blood protein levels. By combining a sample of blood serum with a reagent that turns color in the presence of either the glucose or the protein, and then placing that colored sample in a spectrophotometer, the concentration of the glucose, or protein, can be determined. Your task will be to measure the glucose concentration, or protein concentration, in samples of known concentrations and then use that data to determine the concentration of glucose or protein in a sample of simulated blood serum. As an additional investigation you may choose to measure the glucose concentration of pop, or the protein concentration of milk.

Materials: working with a partner

1. Put on goggles and aprons.
2. Prepare the glucose solution assigned to you by your teacher in a 13x100 test tube. Use Table 1 to determine the amount of standard glucose solution and water to use. Be sure to label the test tube.
3. Using a thin stem pipet add 10 drops of your glucose solution to 2.5 mL of Glucose Reagent in another 13x100 test tube. Mix gently and allow to react for 15 minutes.
4. While your glucose solution is reacting mix 10 drops of the ‘blood serum’ sample provided with 2.5 mL of glucose reagent in another labeled 13x100 test tube. Mix and allow to react for about 15 minutes.
5. While the solution is reacting familiarize yourself with the operation of the TI-82/CBL/Colorimeter. See Colorimeter Handout.
6. At the end of the 15 minute reaction time pour the contents of your glucose solution test tube into a clean cuvette. Do the same for your blood serum sample.
7. Carefully place the first cuvette into the colorimeter, allow the reading to stablize, and record the absorbance in your data table. Repeat this process for the second cuvette.
8. Record the absorbance value for your glucose solution in the class data table as directed by your teacher.
9. All solutions should be washed down the drain with plenty of water. Clean test tubes and cuvettes and return them to their proper place.

Table 1: Preparation of Glucose Solutions

1. Put on goggles and aprons.
2. Prepare the albumin solution assigned to you by your teacher in a 13x100 test tube. Use Table 2 to determine the amount of standard albumin solution and buffer to use. Be sure to label the test tube.
3. Using a clean thin stem pipet add 8 drops of your albumin solution to 3.0 mL of Bradford Reagent in another 13x100 test tube. Mix gently and allow to react for 15 minutes.
4. While your albumin solution is reacting mix 8 drops of the ‘blood serum’ sample provided with 3.0 mL of Bradford reagent in another labeled 13x100 test tube. Mix and allow to react for about 15 minutes.
5. While the solution is reacting familiarize yourself with the operation of the TI-82/CBL/Colorimeter. See Colorimeter Handout.
6. At the end of the 15 minute reaction time pour the contents of your albumin solution test tube into a clean cuvette. Do the same for your blood serum sample.
7. Carefully place the first cuvette into the colorimeter, allow the reading to stablize, and record the absorbance in your data table. Repeat this process for the second cuvette.
8. Record the absorbance value for your albumin solution in the class data table as directed by your teacher.
9. All solutions should be washed down the drain with plenty of water. Clean test tubes and cuvettes and return them to their proper place.

Table 2: Preparation of Albumin Solutions

Analysis:

1. Record class data for both blood components in your data table.
2. Exchange serum data with another group that had the same serum sample.
3. Plot graphs of absorbance versus concentration, using both the glucose and protein solution class data, attach to your report.
4. Obtain the "line of best fit" for each graph.
5. Obtain the equations for the line of best fit and the correlation coefficients.
6. Using the equation for the line of best fit and the absorbance for your blood serum sample, calculate the concentration of glucose and of protein in the serum sample.

Conclusion Questions:

1. How well do your calibration curves match with what we should expect according to Beer’s Law?
2. Write the equations for the line of best fit.
3. Show your calculations for determining the concentration of glucose and protein in your serum sample.
4. What do the glucose and protein concentrations of your serum sample seem to indicate about the person from whom it was taken?
5. Compare your serum sample results with the accepted values provided by your teacher. How well did you do as a clinical chemist?

Colorimeter Handout
Glucose & Protein Analysis
(Adapted from Chemistry with CBL)

1. Make certain that the CBL/colorimeter is ready to use. If not, plug the colorimeter into the adapter cable in Channel 1 of the CBL System. Connect the CBL System to the TI-82 calculator with the link cable using the port on the bottom edge of each unit. Make certain the ends are firmly in place.

    

2. Turn on the CBL unit and the TI-82 calculator. Press and select CHEM. Press then press again to go to the CHEM MAIN MENU.

    

3. Set up the calculator and CBL for the colorimeter.
   • Select 1: SET UP PROBES from the CHEM MAIN MENU.
   • Enter "1" as the number of probes.
   • Select 4: COLORIMETER from the SELECT PROBE menu.
   • Enter "1" as the channel number.
   • Select 1: PERFORM NEW from the CALIBRATION menu.
     
4. You are now ready to calibrate the colorimeter. Use the blank provided by your teacher. The proper blank depends on whether you are doing the glucose assay or protein assay. To correctly use a colorimeter cuvette, remember:

• All cuvettes should be wiped clean and dry on the outside with a tissue.
• Handle cuvettes only by the top edge of the ribbed sides.
• All solutions should be free of bubbles.
• Always position the cuvette with its reference mark facing toward the white reference mark at the right of the cuvette slot on the colorimeter.

To calibrate the cuvette at 0% and 100% transmittance:

• Place the blank cuvette in the cuvette slot of the colorimeter and close the lid. Turn the wavelength knob of the colorimeter to the 0% T position. In this position, the light source is turned off, so no light is received by the photocell. When the voltage reading displayed on the CBL screen stabilizes, press on the CBL and enter "0" (the reference) in the TI-82 calculator.
• Turn the wavelength knob of the colorimeter to the Blue LED position (470 nm) if you are performing the glucose assay, or to the Green LED position (565 nm) if you are performing the protein assay. In this position, the colorimeter is calibrated to show 100% of that particular color of light being transmitted through the blank cuvette. When the displayed voltage reading stabilizes, press and enter "100" in the TI-82 calculator. Leave the wavelength knob of the colorimeter set to the appropriate LED setting for your investigation for the remainder of the experiment.

5. Set up the calculator and CBL for data collection.

• Select 2: COLLECT DATA from the CHEM MAIN MENU.
• Select 1: MONITOR INPUT from the DATA COLLECTION menu

6. You are now ready to collect absorbance-concentration data for your solution. Return to step 7 of the lab procedure.

7. Do not press when you are finished, the other groups may still need to use the colorimeter.

Name __________________

Period ____

1.

2.

3.

4.

5.

Name ___Sample Results_

Period ____

1. See attached graphs. Correlation coefficients indicate good straight line fit, as predicted by Beer’s Law.

2. glucose: absorbance = 0.0312([glucose]) + 0.0233

albumin: absorbance = 0.0391([albumin]) + 0.0279

 

3. glucose: .081 = 0.0312([glucose]) + 0.0233, [glucose] = 1.85 mg/dL x 50 = 92.5 mg/dL

albumin: 0.281 = 0.0391([albumin]) + 0.0279, [albumin] = 6.48 mg/dL x1000 = 6.48 g/dL

4. Glucose is in the normal range, while the albumin is a little high. This would seem to indicate that overall the patient is healthy.

5. Answers will depend on the serum samples provided by the teacher.

The analysis of blood is a very important diagnostic tool for the doctor. Typically the doctor will order several basic tests; usually these include assays for blood components like glucose, protein, creatinine, hemoglobin, calcium, sodium, urea, and potassium. Since the focus of this module is on glucose and protein determination this background will be limited to those two components.

Because glucose is a major energy source for the body it is a very important component of the blood. In order to maintain health it is critical that the glucose level in the blood be kept within certain limits. The body has several mechanisms for accomplishing this. The normal range for glucose is 50 mg/dL - 105 mg/dL. Sustained concentrations outside this range are indicative of some sort of abnormal condition or disease.

Hyperglycemia, or elevated levels of glucose are associated with diabetes. It is estimated that as many as 10 million Americans suffer from some form of diabetes. Hyperglycemia is usually due to reduced insulin production, resulting in the body’s inability to metabolize the glucose. While there are two general types of diabetes; the more severe form, Type I - insulin-dependent diabetes mellitus (IDDM), is the one that most people are aware of. This type of diabetes may lead to the patient developing retinopathy with blindness, kidney failure with the need for dialysis, nerve damage, and circulatory problems possibly leading to heart disease and/or stroke.

Hypoglycemia, or low blood sugar, can be associated with such symptoms as weakness, shakiness, sweating, nausea, and rapid pulse. If serum levels drop to < 20 - 30 mg/dL impairment of the central nervous system can occur. In these cases the symptoms may include: confusion, lethargy, and/or loss of consciousness.

In the case of proteins the composition of the blood is much more complex. There have been over 300 proteins identified in plasma, or serum. But the most concentrated of those proteins is albumin, which constitutes 55% - 65% of the total proteins in human serum. This lab focuses on albumin for that reason. Normal albumin levels are in the range of 3.5 - 5.5 g/dL, and total proteins are in the range 6.0 - 8.5 g/dL.

Protein levels are not as strong a diagnostic tools as glucose, but can indicate certain types of dysfunction. Hyperalbuminemia is only of significance in cases of dehydration. Hypoalbumininemia on the other hand can result from liver disease, tissue damage, malnutrition, nephrotic syndrome, diabetes, lupus, or hypertension. If albumin levels are outside the normal range additional tests are required to pinpoint the dysfunction.

Several special chemicals are needed to carry out the glucose and protein determinations. These chemicals can be obtained from:

Glucose Reagent for glucose determination:

Reagents Applications, Inc.
8225 Mercury CT
San Diego, CA 92111-1203
800-438-6100

Coomassie Brilliant Blue G-250 for Bradford Reagent for protein determination:

Bovine Serum Albumin:

Sigma Chemical Co.
P.O. Box 14508
St. Louis, MO 63178-9916
800-325-3010

Washington State
Essential Academic Learning Requirements

1. The student understands and uses scientific concepts and principles.

    

2. The student conducts scientific investigations.

    

3. The student uses effective communication skills and tools to build and demonstrate understanding of science.

    

4. The student understands how science knowledge and skills are connected to other subject areas and real-life situations.

    

5. The student applies science knowledge and skills to solve problems or meet challenges.

On the following pages are absorbance graphs, suitable for making transparencies. These graphs illustrate some of the basic principles of color and light absorbance. Each of the graphs is described below with suggestions on how to use the graph to illustrate the basic principles.

Transparency 1: This graph shows the absorbance of red food color over the wavelength range 400 - 700 nm. The thing to point out here is that the absorbance peak occurs at about 500 nm. This wavelength corresponds to bluish green light. Since the solution absorbs in the blue-green region what we see is red.

Transparency 2: This graph shows the absorbance of yellow food color over the same range of wavelengths. The thing to point out here is that the solution absorbs strongest at about 400 nm, which is the blue region, causing the solution to appear yellow.

Transparency 3: This graph shows the absorbance of green food color over the same range of wavelengths. One thing to notice here is that it absorbs strongly in two regions, the blue region (the same as the yellow) and at about 625 nm in the orange region. Note that the second peak on this graph corresponds nicely to the main peak on the next graph. This suggests that green food color is a mixture of yellow and blue food color.

Transparency 4: This graph shows the absorbance of green food color over the same range of wavelengths. Point out that the strongest absorbance is at about 600 nm, the yellow region of the spectrum, thus causing the solution to appear blue. It may be helpful to place the green, blue, and yellow spectrums together on the overhead so that students can see the simularities.

Transparency 5: This graph shows the absorbance of the dye used in the glucose reagent over the same range of wavelengths. Point out that the solution appears red because it is absorbing in the greenish-blue region (~500 nm), same as red food color. The 470 nm LED in the colorimeter is choosen because it most closely matches the absorbance peak for the dye.

Transparency 6: This graph shows the absorbance of grape pop over the same range of wavelengths. Have students identify the regions in which it absorbs strongly, and then relate that to the color they observe (absorbs ~500 nm - green, and ~625 nm - orange).

Transparency 7: This graph shows the absorbance of orange pop over the same range of wavelengths. Point out that the orange pop absorbs strongly at ~475 nm (greenish-blue) causing the solution to appear orange

Transparency 8: This graph shows the absorbance of the reagent used for the protein assay over the same range of wavelengths. This graphs is actually a composite of two sets of data. Notice that the reagent alone absorbs very strongly at ~450 nm, but when complexed to the protein the absorbance shifts to ~600 nm. It is because of this peak that we choose the 565 nm LED for the protein measurement.

glucose oxidase

b - D - Glucose + O₂ + H₂O D - Gluconic Acid + H₂O₂

H₂O₂ + Phenol + Reduced Dye Oxidized Dye + H₂O