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Electrochemical-Enzymatic
Determination of Glucose in
Beverages
Last Updated: 5/3/19 by Neil Spinner

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1 Abstract
This laboratory seeks to quantitatively determine glucose concentration in
common beverages.  The method employed is enzyme-catalyzed oxidation of
glucose, quantified by chronoamperometry.

With an inexpensive patterned
electrode and low cost glucose oxidase enzyme, an electrochemical sensor is
constructed to selectively measure glucose in complicated beverages that can
present as analytically-challenging matrices.  This lab briefly discusses conceptual
information about sugars used to sweeten common beverages, enzyme-substrate
interactions, glucose mutarotation, enzyme kinetics, and electroanalysis.
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2 Introduction
Bioanalytical chemistry, a sub-discipline of analytical chemistry, uses quantitative
and qualitative methods of analysis to evaluate biological-based chemical
species.  These biological species are commonly found in complicated matrices
such as blood and may include proteins, drug metabolites, and sugars.
 
In a complicated matrix such as blood, it is difficult to quantitatively determine the
presence and concentration of target analytes.  Therefore, the matrix is often
simplified by chemical separations (e.g., HPLC, GC, electrophoresis) prior to
quantitative determination.
 
Another manner to quantify an analyte among several interferents is by increasing
selectivity.  Many enzymes and substrates function as a lock and key gateway.  The
enzyme catalyzes a reaction only when a very specific substrate binds to its active
site.
 
Such is the case for glucose oxidase and its substrate, β-D-glucose.  Therefore,
despite a complicated matrix of chemical species, an assay based on glucose
oxidase will only have an analytical signal from β-D-glucose (see Figure 1).
 
 Figure 1. Diagram of Glucose Oxidase Enzyme with Substrate ß-D-
glucose and Product H2O2
 
Electrochemical methods are highly sensitive.  Use of electrochemistry with
enzymes as described above adds selectivity.  Therefore, in complicated matrices
(such as common soft drinks, which could contain caffeine, sugars, preservatives,
water, colorings, acids, and other chemicals), electrochemical determination of
the extent of an enzymatic reaction is a sensitive and selective method by which
to determine glucose concentration.
 
The assay described here uses glucose oxidase to catalyze the oxidation of β-D-
glucose to gluconolactone (GNL), with hydrogen peroxide (H2O2) as a byproduct
(see Figure 1).  The hydrogen peroxide is electrochemically detected at a platinum
electrode.  The current from hydrogen peroxide oxidation is calibrated against
standard glucose solutions to construct a calibration curve and determine the
unknown glucose concentration.
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3 Biochemistry Background
The United States Food and Drug Administration (FDA) does not have stringent
policies on nutritional label reporting of sugars (see Figure 2).

On a nutritional
label, the entry for “Sugars” under Total Carbohydrate may include any of the
following naturally occurring or artificially added sugars: fructose, glucose, sucrose,
 and dextrose, and in one of several forms (crystalline, as in sugar crystals, or liquid,
as in high fructose corn syrup).  Any sugars added to the product must be stated
on the ingredients list.  While a general consumer is made aware of added sugars
and total mass of sugar per serving, the customer does not know the
concentration or ratios of the sugars present.

Figure 2. Example of U.S.

Nutrition Facts on Edible
Consumer Goods
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3.1 Types of Sugars

3.1.1 Fructose
Fructose is also called “fruit sugar” as it is the predominant natural sugar found in
plant products such as berries, flowers, and root vegetables (see Figure 3).  For use
in processed foods, fructose is commonly obtained from sugar cane and sugar
beets.

Figure 3. Chemical Structures of Fructose, Glucose, and Sucrose

 
When compared to other sugars, fructose is the sweetest tasting.

 It is a
monosaccharide, the most basic unit of a carbohydrate.  It is absorbed directly

into the bloodstream when consumed by humans and does not elicit a release of
insulin from the pancreas.
 Back To Top

3.1.2 Glucose
Glucose is also a monosaccharide and is the primary source of cellular energy in
plants and animals (see Figure 3).  Commercially, glucose added to foods is most
often sourced from grains, with corn being used most often.  Glucose enters the
bloodstream after processing through the gut and liver.

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3.1.3 Sucrose
Sucrose, commonly referred to as “white” or “table” sugar, is also a
monosaccharide and is a source of cellular energy in plants and animals (see
Figure 3).  Commercially, sucrose added to foods is most often sourced from grains,
with corn being used most often.  Sucrose enters the bloodstream after processing
through the gut and liver.

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3.2 High Fructose Corn Syrup

In recent years, processed food manufacturers use high fructose corn syrup (HFCS)
in lieu of sucrose as a sweetener.  Both consist of glucose and fructose, but HFCS is
less costly due to governmental corn subsidies.  In humans, sucrose is broken down
to glucose and fructose which are processed as monosaccharaides.

 
HFCS is a mixture of the monosaccharide sugars glucose and fructose.  Often,
HFCS is used in processed foods because it is less expensive than sucrose. 
Commonly, perceived sweetness of sugars is based relative to sucrose.  Sucrose
has a rating of 1 on the “sweetness scale,” while glucose and fructose are rated
0.74 - 0.8 and 1.17 - 1.75, respectively.

 
HFCS formulations are prepared and named based on their content of fructose.  A
common blend of HFCS used in soft drinks is HFCS55, which means it contains 55%
fructose and 42% glucose in water (with the balance being maltose).  Based on
relative sweetness, HFCS55 provides sweetness similar to pure sucrose.
 
Consider the sugar content of a cola carbonated beverage.  The label states that
a can with volume 355 mL contains 39 g sugar.  According to the ingredient label,
the sugars are from HFCS.  Therefore, the effective glucose concentration in such a
can would be 16.4 g (0.42 × 39 g = 16.4 g).
 
For quantification of glucose, it is important to realize that the sugar content listed
on a can of carbonated beverage sweetened with HFCS55 is not solely due to
glucose.  In this lab, only glucose, and more specifically only β-D-glucose (the
substrate for the enzyme glucose oxidase) is the analyte of interest.
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3.3 Glucose Mutarotation

Two stereoisomers of glucose are D-glucose and L-glucose.  The D or L designation

with glucose refers to the asymmetric carbon farthest from the aldehyde or keto
 group.  Most naturally occurring sugars are D isomers.  D and L sugars are mirror
images of one another.  D-glucose is the isomer found in nature and in nutritive
sweeteners.  D-glucose can exist in an open chain and closed chain form, the
former being thermodynamically unstable.
 
When a solution of D-glucose is prepared in water, there is a dynamic equilibrium
between the open chain and closed chain forms α-D-glucose and β-D-glucose.

 The cyclic forms of carbohydrates can exist in two forms, α- and β-, based on the
position of the substituent (OH-) at the anomeric center.  The α- and β- forms are
sometimes described as anomers since they are isomers at the anomeric center.
 
Because the open chain is not thermodynamically stable, an equilibrium mixture
between the α-D-glucose and β-D-glucose will result given enough time (see Figure
4).  At room temperature, mutarotation reaches equilibrium in several hours
resulting in a mixture of 36% α-D-glucose and 64% β-D-glucose.
 

Figure 4. Mutarotation Equilibrium Reaction of D-glucose

 
It is important to account for glucose mutarotation when using freshly-prepared
solutions of glucose.  After preparation, solutions must be equilibrated at room
temperature for at least twelve hours before use; otherwise, the ratio of β-D-
glucose and α-D-glucose will be unpredictable and contribute to erroneous
measurements.

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3.4 Glucose Oxidase

Glucose oxidase (GOx) is an enzyme that selectively catalyzes the oxidation of β-
D-glucose to β-D-gluconolactone (GNL) with hydrogen peroxide as a byproduct. 
GOx is a highly selective enzyme.  Even in the presence of nearly identical α-D-
glucose, only β-D-glucose is a substrate for GOx.  Enzyme-substrate interactions act
like a lock and key.  β-D-glucose is like the key while GOx is like the lock as
depicted in the illustration in Figure 1.
 
Overall, the general reaction of interest shows a 1:1 molar relationship between β-
D-glucose and hydrogen peroxide, as shown in Equation 1:
(1)

The bioanalytical assay described here indirectly quantifies glucose by measuring

 The bioanalytical assay described here indirectly quantifies glucose by measuring
the molar concentration of H2O2.  H2O2 is an electrochemically-active chemical
species and can be monitored using electrochemical techniques such as
chronoamperometry.
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3.5 Enzyme Kinetics

The Michaelis-Menten model of enzyme kinetics is perhaps the most straightforward
and well-known model used in biochemistry.  The model is based on a general
kinetic approach for the steps of an enzyme reaction, as shown in Equation 2:

(2)

Enzyme (E), which acts as a catalyst, combines with substrate (S) to form an
enzyme-substrate complex (ES).  This is a reversible equilibrium step with associated
forward (kf) and reverse (kb) rate constants.  The complex then catalytically reacts
to produce product (P) plus enzyme at a specific rate (kcat).
 
From the general reaction (see Equation 2), application of typical first order kinetics
shows that the overall reaction rate is equal to the rate of appearance of product. 
The work of Michaelis and Menten is most recognizable by the reaction rate model
shown in Equation 3:

(3)

where is the reaction rate, [P] is the concentration of enzyme product, is time,
[S] is the substrate concentration, Vm is the maximum rate at saturating
concentration of substrate, and Km is the Michaelis constant, which is equivalent to
[S] when at a rate equal to 1/2Vm.  The reaction rate increases with increasing
substrate concentration, asymptotically approaching its maximum rate when all
enzyme is bound to substrate.
 
The maximum kinetic rate achieved by the system at maximum (saturating)
substrate concentrations is called Vm.  The Michaelis constant (Km) is the substrate
concentration at which the reaction rate is at half-maximum.  Km is inversely
related to the affinity of the substrate for the enzyme.  The value of the Michaelis
constant is dependent on enzyme, substrate, and environmental conditions such
as temperature and pH.

 
The discussion on enzyme kinetics presented above is brief.  It is important to be
aware that the reactions of interest in this laboratory are not simple reactions
driven only by thermodynamic control.
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4 Electrochemical Background
Enzyme-mediated quantification of glucose by chronoamperometry

requires a
basic knowledge of the theory and instrumentation involved.  A wealth of

knowledge exists for both topics.  Here, only a brief overview, sufficient to
understand the methods employed, is given.
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4.1 Chronoamperometry
This lab will use an electrochemical experiment called chronoamperometry.

As
the name implies, current is measured as a function of time while the cell potential
is held constant.  Current at a given time, i(t) (measured in units of amperes), is
directly proportional to concentration, as shown by the Cottrell Equation:

(4)

where n is the number of electrons transferred, F is Faraday’s constant (96485

C/mol), A is the working electrode area (in cm), Do is the diffusion coefficient (in
cm2/s), Co is the concentration (in mol/cm3), and is time (in s).
 
In general, the working electrode is first held at a constant potential, where no
reaction occurs and thus, no charge (current) passes.  Then, the cell potential is
rapidly changed (stepped) to a value that induces faradaic current.

 The current
corresponds to electrons gained or lost during electrolysis (oxidation or reduction).

The value of potential at which the electrolysis occurs is specific to the chemical
system under study.  For each mole of β-D-glucose in a sample, oxidation of the
GOx enzyme produces one mole of gluconolactone and one mole of H2O2.
 
Electrochemically, the H2O2 can undergo a two electron oxidation to form O2 and
H+ (see Equation 5).  H2O2 can be detected at a platinum electrode via oxidation
to O2.  Therefore, after the GOx has catalyzed the conversion of β-D-glucose, the
H2O2 produced will be electrochemically oxidized.  The extent of oxidation
(magnitude of current) will be measured by chronoamperometry and compared
against a calibration of several standard solutions to determine the unknown
concentration of β-D-glucose in the sample.

(5)

As evident from the electrochemical half-reaction (see Equation 5), H2O2 is

oxidized to O2 between 600 to 700 mV vs. an Ag/AgCl reference electrode.

To
oxidize hydrogen peroxide produced by the action of glucose oxidase, the cell
potential is stepped from an initial resting potential (where neither reaction occurs
nor electrons flow) to a potential more positive than 600 to 700 mV.  At 900 mV, any
H2O2 near the electrode surface will be oxidized.  For each mole of H2O2 oxidized,
two electrons will flow into the external circuit (measured as current in the
potentiostat).  After a specified amount of time, current is measured and verified
against a calibration curve to determine the concentration of β-D-glucose in the
sample.  β-D-glucose is converted to gluconolactone and peroxide by glucose
oxidase (see Figure 5).  The peroxide generated is oxidized at the platinum
electrode (slant lines).  Current is monitored and related to calibration.
 
 Figure 5. Electrode Reaction for Glucose Detection
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4.2 Electrochemical-to-Biochemical Data Translation

In this experiment, you measure the rate of enzymatic reaction through
electrochemical oxidation of the enzymatic byproduct, which is an indirect
measure of enzyme turnover.  Thus, you must translate the electrochemical rate
(current vs. time) to reaction rate (concentration vs. time).  Algebraically
rearranging the Cottrell equation (see Equation 4) provides a fairly simple path by
which to accomplish this in a spreadsheet, as shown in Equation 6:

(6)

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4.3 Electrochemical Instrumentation

There are two main components of the electrochemical instrumentation used in
this experiment: the potentiostat and the electrodes.
 
WaveNow USB Potentiostat

with AfterMath Data Organizer Software

Compact Voltammetry Cell

Screen-printed Platinum Electrodes

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4.3.1 Electrodes
Electrodes used in this experiment are called the working, counter, and reference
electrodes.  The electrochemical reaction of interest occurs at the working
electrode.  The working electrode is a material that is a sink for electron transfer. 
The reference electrode has its own fixed redox potential and serves as a standard
reference for cell potential.  Current should not pass through a reference
electrode; thus, the counter electrode (or auxiliary electrode) is the third electrode
in the system.  Current passes through the counter electrode to balance a change
in cell potential due to the reaction at the working electrode.
 
For this experiment, the platinum screen-printed electrode conveniently contains
all three electrodes (see Figure 6).
  

Figure 6. Pine Research Platinum Screen-Printed Electrode

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5 Chemicals
For all solutions, consult with your instructor.  Some may have been prepared in
advance.  For other solutions, they may be prepared from a higher concentration
stock solution that has been provided.  The volumes listed are for one student or
one group of students working together.  The water used for these solutions should
be distilled and deionized.  Such water typically has a resistivity of 18 MΩ cm.
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5.1 Sodium Acetate Buffer (1.0 M)

Sodium acetate is abbreviated NaOAc.  Calculate the appropriate mass of
NaOAc to prepare 100 mL of a 1.0 M solution.  Add this mass to a 100 mL
volumetric flask and add about 70 mL distilled water.  Then, add 4.2 mL
concentrated hydrochloric acid (36.5% - 38% v/v).  Swirl to completely dissolve,
then dilute to the mark with distilled water.  Label this solution as 1.0 M NaOAc
Buffer.

CAUTION: Concentrated hydrochloric acid is highly corrosive and

causes severe burns to eyes, skin, and respiratory tract.  Reaction with
 water produces excessive heat (exothermic); therefore, always add
acid to water and never water to acid.  Use all appropriate personal
protective equipment.

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5.2 Sodium Acetate Buffer (50 mM)

Prepare 500 mL of a 50 mM NaOAc buffer by diluting the 1.0 M NaOAc buffer
previously prepared (HINT: M1V1 = M2V2).  Label this solution as 50 mM NaOAc
Buffer.
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5.3 Glucose Oxidase Solution (100 IU/mL)
Dissolve 50 kU (fifty thousand IU) of glucose oxidase in 500 mL of the previously-
prepared 50 mM NaOAc Buffer.  Stir gently with a glass rod to avoid creating
bubbles.  Keep refrigerated for storage and warm to room temperature before
use.  Label this solution as 100 IU/mL Glucose Oxidase Stock.

INFO: Enzyme concentrations are often expressed in units of IU

(International Units).  One IU is the amount of enzyme that will produce
 1 µmol of product per minute (or, that will consume 1 µmol of reactant
per minute).  Prepare this solution based on the concentration stated
on the bottle.

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5.4 Glucose Stock Solution (100 mM)

Calculate the appropriate mass of D-(+)-glucose (molecular weight = 180.16
g/mol) to prepare 100 mL of a 100 mM solution.  Add this mass to a 100 mL
volumetric flask and add about 75 mL of the 50 mM NaOAc Buffer and swirl to
dissolve completely.  Then, dilute to the mark and label as 100 mM Glucose Stock
Solution.

NOTE: Prepare this solution at least 12 hours in advance.  This allows

mutarotation of the α- and β- optical isomers of glucose to reach
 equilibrium.  Remember, only β-D-glucose is a substrate for the enzyme
glucose oxidase.

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5.5 Beverage Sample Stock Solution

Prepare a stock beverage sample.  Select any glucose-containing beverage. 
Beverage selection may affect results.  Some sweetened beverages contain little
glucose despite having a high content of “sugars” listed on the nutritional label. 
Record the nutritional label data and ingredients list for each beverage sample
into a notebook.
 
If the beverage is carbonated, decarbonate it prior to use (i.e., remove all gas
bubbles).  This is accomplished by placing the soft drink in a plastic bottle and
sealing the cap, shaking for 20-25 seconds, and then slowly opening the cap to
release CO2.  Repeat this procedure until no observable carbonation is present.
 
Mix 0.8 mL pure beverage with 0.5 mL 1.0 M NaOAc Buffer and 8.7 mL water.  Label
this solution as Beverage Stock Solution.
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5.6 Beverage Sample Test Solution

Test beverage samples should be analyzed immediately after preparation The
 Test beverage samples should be analyzed immediately after preparation.  The
preparation for the beverage solution tested will be given in the experimental
section.  Record nutritional information label content and any sugars listed in the
ingredients label for any beverage tested.
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5.7 Glucose Calibration Standards

Prepare a series of at least five glucose standards.  Make these standards from the
previously-prepared 100 mM Glucose Stock Solution and 50 mM NaOAc Buffer (see
Table 1).  It will be easiest to prepare each sample in separate 20 mL disposable
vials (e.g., scintillation vials,

which fit the compact voltammetry cell cap). 
Properly label each standard prepared.
 

[Glucose]
100 mM Glucose Stock
50 mM NaOAc Buffe
Standard #
(mM) (mL) (mL)

1 0 0.0 5.0

2 5 0.5 4.5

3 10 1.0 4.0

4 15 1.5 3.5

5 20 2.0 3.0
Table 1. Glucose Standard Preparations
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6 Experimental
In this lab, the concentration of glucose in a beverage sample will be obtained by
oxidizing an enzymatic byproduct of glucose oxidase, hydrogen peroxide.  After
you have made the solutions, proceed to the instrumental setup.  The analysis
portion involves construction of a calibration curve from glucose standards and the
analysis of a beverage sample as compared to the calibration.  Read through the
experimental section completely before proceeding.
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6.1 Setup
The following instrumentation supplies will be needed to perform this experiment:
the Pine Research WaveNow potentiostat

and USB cable, platinum screen-
printed electrode,

compact voltammetry cell,

and the previously-prepared
solutions.  A stopwatch must also be used to accurately ensure all samples have
reacted with enzyme for the same duration of time.
 
1. Turn on the potentiostat and connect it to a personal computer using the USB
cable.  Use the AfterMath software application to control the potentiostat.
2. Connect the cell cable to the potentiostat using the end terminating with an
HD-15 connector.  The other end of the cable will be a mini-USB-style
connector.

3. Obtain one platinum screen-printed electrode.  This electrode has a working

electrode in the center that has a 2.0 mm diameter (see Figure 6).  Compute
 the surface area of the working electrode (in cm2) and record the result.
4. From the Experiments menu in AfterMath, select “Chronoamperometry
(CA)…”  Notice, this will create a new Experiment Node in the archive structure
on the left panel of the screen (see Figure 7).
 

Figure 7. Chronoamperometry Experimental Parameters

 
5. Select the appropriate potentiostat from the drop-down menu at the top of
the parameter entry screen (e.g., Pine WaveNow).
6. Perform a CA experiment consisting of the following parameters: 300 mV
induction period potential for 2 s; 900 mV forward step potential for 29 s; 900
 mV relaxation period potential for 0 s; initial current range = 200 µA with
Autorange Off; and 2900 intervals (see Figure 7).
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6.2 Glucose Calibration

The enzyme-glucose reaction is kinetically-limited; therefore, it is important to
prepare and analyze each standard before moving on to another.  Work
systematically and in sequence, from lowest to highest concentration of glucose. 
Analyze each of at least five standard glucose samples, given in Table 1, in the
following manner:
 
1. Rinse the electrode with deionized water and dab dry with a delicate wipe. 
Set aside.
2. Obtain a standard sample (5 mL of solution in a 20 mL threaded vial).
3. Add the 5 mL Glucose Oxidase Solution to the test solution.  Shake well, and
immediately start a timer.
4. Allow the enzyme to react for 5 minutes.
5. After 5 minutes, insert the platinum screen-printed electrode into the hand grip
(see Figure 8).
 

Figure 8. Assembly of the Compact Voltammetry Cell

 

6. Insert the hand grip, which is connected to the electrode, into the cap (see
Figure 8).
 7. Connect the USB-style cell cable to the grip.  The correct connector is to the
LEFT of the electrode face of the screen-printed electrode card when facing
downward (see Figure 8, right photo).
8. In AfterMath, highlight the CA Parameters icon in the tree structure on the left
(that you created above).  Click the “Perform” button to begin the
chronoamperometry experiment.
9. After the experiment completes, right click the experiment node in the left
panel of AfterMath (e.g., CA Experiment (00##)) and rename it to indicate the
glucose concentration used.
10. Remove the grip and cap from the vial.  Dispose of the waste appropriately. 
Repeat these steps for each calibration standard until you have obtained data
for all five standards.
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6.3 Beverage Analysis

To a clean 20 mL threaded vial, add 5 mL Beverage Sample Stock Solution and 5
mL of the Glucose Oxidase Stock.  Mix well, and start a timer.
 
Allow the enzyme to react for 5 minutes.  Perform chronoamperometry on the
sample as with the glucose calibration.  The measurement can be made in
triplicate for statistical validation of the data.
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7 Data Analysis
The resultant data from a chronoamperometry experiment, called a
chronoamperogram, is a plot of current vs. time.  The current arises from the
electrochemical oxidation of H2O2 (see Equation 5 and Figure 5).  From Equation 5,
the measured current is proportional to the concentration of peroxide.  From the
overall enzyme reaction, one mole of glucose is oxidized for every mole of
peroxide produced and subsequently electrochemically oxidized.
 
To maintain assumptions for the Michaelis-Menten model of enzyme kinetics
(discussed in most biochemistry textbooks),

a common enzyme assay is to vary
substrate concentration and measure the reaction rate.  In this lab, current is a
direct measure of the kinetic rate (the chronoamperogram gives current as a
function of time for the oxidation of hydrogen peroxide, which is in molar
equivalency with substrate).
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7.1 Determine Cottrell Current at 8 s

In addition to recording data in your notebook, open a spreadsheet and create
an organized system to analyze data.
 
For each glucose standard, review the corresponding chronoamperogram in
AfterMath.
 
1. Expand the appropriate experiment node and select the “Current” node.
2. In the graphical data window in the right panel of AfterMath, right-click on the
data trace.  From the menu, select Add tool and then Crosshair.
 3. A large plus sign will lock to the data trace.  The crosshair tool will report the x
and y values for a certain point on the data, in this case expressed as time (x-
intercept) and current (y-intercept).  With the mouse, move the crosshair to the
data point at a time of 8 seconds.  You may have to use the plot tools (such as
zoom in) to more easily select exactly 8 seconds (see Figure 9).
 

Figure 9. Determination of Cottrell Current at 8

 
4. Record the current at 8 seconds.
5. Repeat this process for each glucose standard.
6. In the spreadsheet, enter concentration in one column and current at 8
seconds in the next.
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7.2 Data Transformation
As described previously (see Section 4.2), convert current to reaction rate (see
Equation 6).  HINT: current is in amperes in the Cottrell Equation, and is a
3
concentration in units of mol/cm .  Use formulas in your spreadsheet to avoid
simple mathematics errors.
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7.3 Create Michaelis-Menten Plot

Create a plot of reaction rate (y axis) vs. glucose concentration (x axis).  Plot the
data as a series of points without a line connecting the points (see Figure 10).
 

Figure 10. Michaelis-Menten Plot for Glucose Oxidase

 
As discussed in the Biochemistry Background, the data in the calibration plot
should not be linear across the range tested.  Lack of linearity is due to the two
limiting factors of the glucose oxidase enzyme system: 1) the amount of substrate
(β-D-glucose) available; and 2) the maximum turnover rate of the glucose oxidase
enzyme.
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7.4 Create Lineweaver-Burk Plot

Transform the data used in the previous plot to the Lineweaver-Burk (double
reciprocal) form.  Plot 1/ on the y axis and 1/[S] on the x axis.  The data should
now appear linear.  Perform a linear regression analysis and display the equation of
the best-fit line (see Figure 11).  Note the slope and intercept and consider their
proper units.

 
 Figure 11. Lineweaver-Burk (Double Reciprocal) Plot for Glucose Oxidase
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7.5 Determine β-D-Glucose Concentration from Analysis in Beverage

Determine the peroxide current for each beverage tested, as discussed previously
(see Sections 7.1 and 7.2).  Determine β-D-glucose concentration in each
beverage by using the calibration curve in linear format.  Report the concentration
(HINT: account for all dilutions).
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7.6 Determine Kinetic Parameters

From the slope and intercept of the linear regression performed on the double
reciprocal plot, determine Vm and Km and record these values (HINT: solve as a
system of two equations with two unknowns).

(7)

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8 Report Questions
1. Compare glucose concentration as determined by this method to the
nutritional label on the beverage.  Do the values agree?  Why or why not?  Are
there clues in the ingredient list that support your observations?
2. If a current measurement by chronoamperometry was taken immediately after
glucose oxidase was added to the sample, how would that affect the
concentration measured by chronoamperometry?
3. Why are all solutions in this lab in a buffered NaOAc solution? (HINT: consider
general properties of enzymes)
4. How should current vary with peroxide oxidation?
5. This experiment used a 2 mm disk electrode.  If a working electrode with a 5
 5. s e pe e used a d s e ec ode.  a o g e ec ode a5
mm disk was used instead for the glucose calibration, discuss the change
expected in current response.  What is the proportionality between electrode
area and current?
6. State the values of Vm and Km, with the appropriate units.  Show the
calculation of Vm and Km from the linear calibration data.
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9 References
U.S. Government.  Title 21 – Food and
Drugs.  https://www.govinfo.gov/content/pkg/CFR-2008-title21-vol2/pdf/CFR-
2008-title21-vol2-part101-subpartA.pdf.

Ventura, E. E.; Davis, J. N.; Goran, M. I.  Sugar Content of Popular Sweetened

Beverages Based on Objective Laboratory Analysis: Focus on Fructose
Content.  Obesity, 2011, 19(4), 868–874.

Hanover, L. M.; White, J. S.  Manufacturing, composition, and applications of

fructose.  The American Journal of Clinical Nutrition, 1993, 58(5 Suppl), 724S-
732S.

Nelson, D. L.; Cox, M. M.  Lehninger Principles of Biochemistry, 6th ed. W.H.

Freeman {&} Company, 2012.

Perles, C. E.; Volpe, P. L. O.  A Simple Laboratory Experiment To Determine the

Kinetics of Mutarotation of D-Glucose Using a Blood Glucose Meter.  J. Chem.
Educ., 2008, 85(5), 686.
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Software Applications
 Application notes discuss practical aspects
Detailed information about our Software,
of conducting specific experiments.
which includes AfterMath and retired
PineChem.

Theory Product Specifications

Fundamental electrochemical theory
presented in a brief and targeted manner. Review complete product specifications
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