This document describes an internally calibrated electrochemical continuous enzyme assay (ICECEA) for determining enzyme activity. The ICECEA allows for the concurrent calibration and enzyme assay in the same solution and at the same electrode. It provides current-time traces that are used to calculate the calibration slope and assay slope to determine enzyme activity levels. Examples are provided for alcohol dehydrogenase, lactate dehydrogenase, and glucose oxidase assays. The document also describes using the ICECEA to detect myeloperoxidase activity and inhibitors using hydrogen peroxide and sodium cyanide substrates. Finally, it outlines a protocol for immobilizing myeloperoxidase on silica-chitosan particles through glutaraldehyde coupling.

1. Electrochemical Enzyme Assay
Octobre 2021

2. Waldemar Gorski, Ph.D.
PROFESSOR
University of Texas at San Antonio
Office: BSE 1.104M
Phone: (210) 458-4961
Email: waldemar.gorski@utsa.edu
Internally calibrated electrochemical
continuous enzyme assay(ICECEA),
(patent pending)

4. The ICECEA was performed in the
constant-potential amperometric
mode in a stirred solution of
enzyme’s substrate.
After recording a baseline current,
the known aliquots of a product (or
reactant) of enzymatic reaction
were added to the solution in order
to record the current steps that
were used to calibrate the assay.
This was followed by the addition
of assayed enzyme in order to
trigger an enzymatic reaction and
record an angled current− time
(I−t) segment.
CS= calibration slope CS
AS= assay slope AS
5 min experiment

5.  enzyme substrate + reactants products
 The initial rate reflects the enzyme activity, which by definition is expressed
as the amount of enzyme’s substrate that is converted to a product per unit
time (1 U = 1 micromole min−1). The determination of U is based on
measuring either the formation of a product or consumption of a reactant of
the enzymatic reaction 1 over time. The unique feature of the ICECEA is that
the preassay calibration and actual enzyme assay are performed consecutively
in the same solution and at the same electrode.
Principle of ICECEA
Enzyme

6. Alcohol dehydrogenase
ICECEA current−time traces (E = 0.40 V)
recorded at a
glassy carbon/CNT film electrode in a stirred
solution of 0.56 Methanol in pH 8.80
pyrophosphate buffer solution (25 °C). The
calibration I−t steps are due to the additions
of 1.0 μM aliquots of NADH. The angled I−t
segment was recorded after the addition of
3.74 U L−1 alcohol dehydrogenase.
Ethanol+ NAD acetylaldehyde + NADH
alcohol
dehydrogenase

7. Lactate dehydrogenase
ICECEA amperometric traces (E = 0.40 V)
recorded at a glassy carbon/carbon nanotubes
film electrode in a stirred solution of 2.30 mM
sodium pyruvate in pH 7.50 phosphate buffer
solution (37 °C). The calibration I−t steps are
due to the additions of 25.0 μM aliquots of
NADH. The angled descending I−t segment
was recorded after the addition of 10.0 U L−1
lactate dehydrogenase.
Pyruvate + NADH Llactate + NAD+
lactate dehydrogenase

8. Glucose oxidase
ICECEA amperometric traces (E = 0.60
V) recorded at a
platinum electrode in a stirred solution of
0.10 M glucose in pH 5.10 acetate buffer
(35 °C). The calibration I−t steps are due
to the additions of 2.0 μM aliquots of
hydrogen peroxide. The angled I−t
segment was recorded after the addition of
19.9 U L−1 glucose oxidase.
B-D-Glucose + O2 + H2O Glucono-1,5 lactone + H2O2
glucose oxidase

9. Fig. 2. ICECEA amperograms (–0.20 V)
recorded (A) at N-CNT film electrode in
10 mM SCN− solution that was stirred at
155 rpm. Current steps before 200 s are
due to addition of three calibrating
aliquots each yielding 7.0 μM H2O2 in a
solution. At 200 s, a MPOcov film
electrode loaded with (a) 0.29, (b) 0.58,
(c) 1.2, (d) 2.3, and (e) 3.9 mU MPO was
dipped into a solution; (B) at MPOcov film
electrode (loaded with 2.3 mU MPO) in a
background electrolyte solution. At 200 s,
an aliquot was added to yield 10 mM
SCN− in a solution that was stirred at (d)
155 rpm, and (d1) 380 rpm. MPOcov films
contained N-CNT. Background
electrolyte, pH 7.40 PBS.
MPO

11. Background-corrected cyclic
voltammograms recorded in a 1.0
mM H2O2 solution at (1) MPOcov,
and (2) MPO-free film electrode that
contained (A) N-CNT, (B) MWCNT,
(C) SWCNT, and (D) no CNT. The
MPOcov films were loaded with 1.2
mU MPO. Original voltammograms
are shown in Figs. S1–S4.
Background electrolyte, pH 7.40
PBS. Scan rate, 50 mV s−1

12. SCN Amperometric detection
Amperometric traces (–0.20 V) recorded
at (1) MPOcov, and (2) MPOaff film
electrodes in a stirred solution (155 rpm)
that was spiked with 11 SCN− aliquots to
yield 1, 5, 10, 10, 25, 25, 25, 50, 100, 250,
and 500 μM SCN− in a solution. The MPO
films were loaded with 1.2 mU MPO and
contained N-CNT. Inset: Calibration plots
based on amperometric traces.
Background electrolyte, pH 7.40 PBS
containing 1.0 mM H2O2.

13. MPO inhibitor detection
Cl + H2O2 H2O + HClO
MPO
H2O2 on the surface of platinum electrode
(E = 600 mV (vs. Ag/AgCl)) . Or
CNT/Glassy carbon Electrode at -200 mV
(vs. Ag/AgCl)) .
 HClO on the surface of platinum
electrode (E = 150 mV (vs. Ag/AgCl)) .

15. SiO2/ Chitosan/ Glutaraldehyde/MPO

17. Protocol for silica- chitosan- glu-MPO
 Prepare a 1 M acetic acid 100 ml by dissolving 6.05 g acetic acid in
distillated water for 100 ml final volume .
 Prepare chitosan 5% by dissolving 2.5 g chitosan in 47.5 g acetic
acid 1M , 90 0C, and stirring for 2h, then filter by 0.45μm syringe
filter.
 Impregnated 0.5 gr silica gel in 10 ml chitosan 5%, stirring
overnight.
 Centrifuge the mixer.

18. • Incubate Impregnated silica particle (0.5 g) in 5 mL 0.1 M acetate buffer
(pH 4.0) containing 2.5% glutaraldehyde in an orbital shaker at 25 ° C and
200 rpm for 1 h. The excess glutaraldehyde will be removed by washing with
distilled water
• Dissolve 125 μl of MPO solution (800 μl / μg) in 2 ml 0.1 M PB, 300 mM
NaCl pH=7.4. add 0.2 g activated silica particles and allow the solution to
rotate for 2 h at room temperature.
• Centrifuge the mixer
• Use the upper solution for unreacted MPO activity measurement according to
MPO assay protocol.

19. • Wash Silica attached MPO with 0.1 M PBS pH=7.4 two times.
• Mix 0.2 gr of Silica attached MPO with 0.1 M PBS pH=7.4, containing 0.1M
cyanoborohydride for 30 min in 2ml final volume.
• Wash and mix with 0.1 M PBS pH=7.4, containing 15% ethanolamine (for increasing
the activity of enzyme) for 2 h in 2ml final volume.
• Homogenize 0.2 gr silica attached MPO solution with 2ml 0.1 M PBS pH=7.4, and
take 50ul of it for measuring the chlorination and peroxidation activity according to
step 9 and 10.
Ref: 1. Reactive & Functional Polymers 66 (2006) 682–688
2. Sensors & Actuators: B. Chemical 331 (2021) 129469