Talanta 50 (2000) 1275 1281

www.elsevier.com/locate/talanta

The electrogenerated chemiluminescent behavior of hemin

and its catalytic activity for the electrogenerated
chemiluminescence of lucigenin
Guo Nan Chen a,*, Lin Zhang a, Rong Er Lin b, Zhen Cong Yang a,
Jian Ping Duan a, Hong Qing Chen a, D. Brynn Hibbert c
a

Department of Chemistry, Fuzhou Uni6ersity, Fuzhou, Fujian, 350002, China

b
Bureau of Commodity Inspection, Samming, Fujian, 365000, China
c
School of Chemistry, The Uni6ersity of New South Wales, Sydney, 2052, Australia
Received 24 March 1999; received in revised form 28 July 1999; accepted 3 August 1999

Abstract
The electrogenerated chemiluminescent (ECL) behavior of hemin at a platinum electrode in the alkaline solution
has been investigated in detail. Under the optimum conditions the linear response range of hemin is 1.0 10 5
1.0 10 8 g ml 1, the detection limit was 1.0 10 8 g ml 1, and the relative standard derivation for 110 7 g
ml 1 hemin was 2.8%. It has been also found that hemin would catalyze the ECL of lucigenin at a platinum electrode
in a neutral solution in the presence of hydrogen peroxide, the catalytic ECL intensity was linear with the
concentration of hemin in the range of 1.0 10 14 1.010 10 g ml 1. IgG labeled with hemin was used to
examine the ECL catalytic activity of hemin after conjugating to protein, and the results showed that hemin retained
ECL catalytic activity when conjugated to protein. 2000 Elsevier Science B.V. All rights reserved.
Keywords: Hemin; Electrogenerated chimluminescence; Lucigenin; IgG

1. Introduction
Recently nonradioactive labels such as enzymes, stable free radicals and chemiluminescent
(CL) reagents have been used in attempts to overcome the intrinsic shortcoming of radioimmunoassay. Of these labels an enzyme is a
promising alternative label, as an enzyme is a
* Corresponding author. Fax: +86-591-3713866.
E-mail address: gnchen@fzu.edu.cn (G. Nan Chen)

catalyst and it can be determined with high sensitivity by various methods. For example,
horseradish peroxide (HRP) is an effective catalyst for the luminol-H2O2 CL system that has
been widely used [13]. However, in enzyme immunoassay, the steric hindrance of macromolecular coumpound and the instability of high
molecular labeling enzymes would affect the antigenantibody reaction, Therefore, some mimetic
enzymes have been developed to be used as a
substitute for natural enzyme in analytical appli-

0039-9140/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved.
PII: S 0 0 3 9 - 9 1 4 0 ( 9 9 ) 0 0 2 4 0 - 4

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G. Nan Chen et al. / Talanta 50 (2000) 12751281

cations. Various metalloporphyrins have been

used as mimetic peroxidase in fluorescence and
CL analysis [4 6].
Hemin is a well-known natural metalloporphyrin compound. The difference between hemin
and synthetic metalloporphyrin is that hemin has
no substituted group at positions 9, 10, 11, 12,
and it has two carboxyl groups, which can condense with a hydroxyl or amino group in protein.
Ikarlyama [7] developed a method termed chemiluminescent catalytic immunoassay in which he
used hemin as the labeling agent to label human
serum albumin (HAS), which would catalyze the
CL reaction between luminol and H2O2. However,
no attention has been paid to the ECL behavior
of hemin. We have recently found that hemin
gave a reproducible ECL signal at a platinum
electrode in NaOH-KCl solution when a triangular pulse voltage was applied. We also found that
hemin could catalyze the ECL reaction of lucigenin at a platinum electrode by using KCl as the
supporting electrolyte.
In this paper, the ECL behavior and mechanism of hemin itself at a platinum electrode in
alkaline solution have been investigated in detail,
and the catalytic characteristics of hemin for the
ECL reaction of lucigenin at a platinum electrode
in a neutral solution have also been discussed.
IgG labeled with hemin was used to examine the
catalytic activity of hemin after conjugating to
protein.

2. Experimental

2.1. Apparatus
A Shimadzu RF-540 spectrofluori meter, a
Perkin-Elmer Lambda 9 Spectrophotometer and a
BAS-100 Electrochemical Analyzer (Bioanalytical
System, Purdue, In, USA) were used. ECL experiments were performed with a system made in our
laboratory, which consisted of an HFC-Chemiluminescent Detector, a potentiostat, an electrochemical cell and a recorder. A block diagram of
this system has been shown in the literature [8,9].
The details of the HFC-I CL Detector have been
given previously [10]. The potentiostat used was

XHD-I (Precision Instrumental Company of Xiamen University, Xiamen, China) it mainly included a waveform generator, which could
perform linear, triangular voltage, square wave
and double-step pulse voltage sweep. ECL experiments utilized a platinum plate working electrode(4 6.5 mm, sealed in glass) in a
conventional three electrode cell configuration.
The auxiliary electrode was a platinum ball electrode (d= 2.2 mm, sealed in glass), and the reference electrode was an AgAgCl electrode. The
photomultiplier tube, GDB-413 (Nanjing Electron
Tube Works) with a detection range of 300700
nm was housed directly in front of the electrochemical cell.

2.2. Reagents
Solution of hemin: 10.0 mg of hemin (Sigma)
was dissolved in 5 ml of 0.2 mol l 1 NaOH, and
was then diluted to 100 ml to give a stock standard solution containing 1 10 4 g ml 1 of
hemin. This solution was diluted to the required
concentration with water.
Solution of lucingenin: 52.2 mg of lucigenin
(Aldrich) was dissolved in 100 ml of water to
obtain a stock standard solution containing 1.0
10 3 mol l 1 lucigenin. This solution was diluted
to the required concentration with water.
IgG was obtained from Shanghai Biological
Product
Institute,
1-ethyi3(3dimethylaminopropyl) carbodiimide hydrochloride (EDC) was
obtained from Sigma. All the other reagents used
were analytical reagents or better and all water
used were doubly distilled in a fused-silica apparatus.

2.3. Procedure
2.3.1. Measurement of ECL of hemin
The cell filled with 10 ml solution containing
hemin, 0.4 mol l 1 KCl and 0.03 mol l 1 NaOH
was placed in the detector chamber. A potential
was then applied to the working electrode and the
ECL signal was recorded. After each measurement, the working electrode and the auxiliary
electrode were exchanged and electrolyzed at 1.0
V for 1 min in 0.5 mol l 1 HNO3 before the next
measurement.

G. Nan Chen et al. / Talanta 50 (2000) 12751281

2.3.2. Measurement of ECL of lucigenin

catalyzed by hemin
The cell filled with 10 ml solution containing
hemin, 0.1 mol l 1 KCl, 1 10 6 mol l 1 lucigenin and 110 6 mol l 1 H2O2 was placed in
the detector chamber. The subsequent procedure
was the same as measurement of ECL of hemin.
2.3.3. Preparation of hemin-labeled IgG
Thirteen milligrams of hemin was dissolved in 3
ml of water, 27.2 mg EDC was then added. The
mixture was incubated for 10 min at room temperature with stirring. A mass of 10.2 mg IgG in
4 ml of water was added to the above mixture and
the mixture was allowed to stand at room temperature for 10 h with stirring. Ten milligrams of
glycine was added to stop the reaction. The reaction mixture was dialyzed 24 h at 4C against the
pH 7 phosphate buffer solution (the buffer solution was changed five times during dialysis). The
resulting labeled protein was chromatographed on
a Sephadex G-25 column (diameter, 17 mm; l, 400
mm), and eluted with 0.1 mol l 1 phosphate
buffer solution (pH 7). All fractions eluted from
the column (3 ml) was used for spectrophotometric and ECL studies.

3. Results and discussion

3.1. The ECL beha6ior of hemin

3.1.1. Selection of the conditions for ECL
reaction of hemin
The preliminary investigation showed that
hemin gave ECL in alkaline KCl solution at the
platinum electrode when triangular voltage was

1277

applied. Considering that some oxidants and reductants would enhance the ECL of some metallic
complex system [11,12], the ECL behavior of
hemin in different media: KCl-NaOH, KClNaOH-H2O2, KCl-NaOH-NH4S2O8, KCl-NaOHNa2C2O4 solutions were investigated in detail. The
results are shown in Table 1. It can be seen from
Table 1 that the presence of the oxidants and
reductants would inhibit the ECL of hemin, and
the inhibition was increased with the concentration of oxidants and reductants. We attempt to
use a buffer solution containing carbonate, phosphate and acetate to control pH, however, we
found that all these anions would decrease the
ECL of hemin. Therefore, KCINaOH system
was used for the subsequent investigation.
The effect of NaOH concentration on the ECL
intensity of hemin was investigated, and the result
showed that the ECL intensity was increased with
NaOH when its concentration was lower than
0.02 mol l 1. When concentration of NaOH was
higher than 0.02 mol l 1, the ECL intensity
reached maximum and became constant. Thus,
0.03 mol l 1 NaOH was selected for subsequent
studies.
KCl was used as the supporting electrolyte. The
ECL intensity of hemin did not change much
when the KCl concentration was varied between
0.30.5 mol l 1, when KCl was lower than 0.3
mol l 1, the ECL intensity was decreased greatly,
and after 5 10 2 mol l 1, hemin almost did not
give ECL. Meanwhile, we also found that KCl
itself gave an ECL background; 0.4 mol l 1 KCl
was used for subsequent investigation.
Square, triangular and symmetric double step
pulse voltages were selected to examine the ECL
behavior of hemin at the platinum electrode.

Table 1
The ECL intensity of hemin in various media (hemin = l.0106 g ml1)a
KCl-NaOH-H2O2

KCl-NaOH-(NH4)2S2O8

KCl-NaOH-Na2C2O4

ECL system

KCl-NaOH

A1

A2

B1

B2

C1

C2

IECL

21

14.5

9.2

12.0

8.0

7.0

9.0

a
NaOH, 0.03 mol l1; KCl, 0.4 mol l1; A1: H2O2, 3.0103 mol l1; A2: H2O2, 3.0102 mol l1; B2: (NH4)2S2O8 = 1.0
103 mol l1; B2: (NH4)2S2O8 = l.0103 mol l1; C1:Na2C2O4, 1.0104 mol l1; C2: Na2C2O4, 1.0103 mol l1.

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G. Nan Chen et al. / Talanta 50 (2000) 12751281

Table 2
Optimum conditions for the ECL of hemin
Electrochemical parameters

Solution conditions (mol l1)

Pulse waveform
Pulse amplitude
Static stand time
Pulse period

Triangular
1.5 V
15 s
8 ms

Hemin gave only a very weak ECL under symmetric double-step and square pulse voltages, but
the ECL of hemin was strong when triangular
pulse voltage was applied. Therefore, the triangular pulse voltage was selected for subsequent
studies.
A triangular pulse voltage was selected to examine the relationship between the potential and
ECL intensity. It was found that when the potential was lower than 1.0 V, the ECL signal was
very weak, above which the intensity increased
with increasing potential. The intensity reached a
maximum and constant value above 1.5 V. Thus a
1.5 V triangular pulse voltage was selected for
subsequent studies.
The effect of pulse duration of the range 0.22
22 ms was tested, and the results showed that the
ECL intensity of hemin increased with increasing
pulse duration; when the pulse period was 8.0 ms,
the intensity was strongest and the peak shape
was better. For a fast ECL reaction system, after
each measurement, the solution should be stood
for some time to let the diffusion layer around the
electrode return to its original state, thus better
reproducibility could be obtained. It was found
that a 15 s static time was enough to give repeatable measurement. The optimum conditions for
the ECL of hemin used in subsequent studies is
shown in Table 2.

3.1.2. Linear response range, detection limit and

precision
Under the above optimum conditions, the linear response range was 1 10 5 2 10 8 mol
l 1 hemin. The minimum detectable concentration of hemin (defined as the concentration which
could be detected for a signal-to-noise ratio of 3)
was 1 10 8 mol l 1, and the relative standard

NaOH
KCl

0.03
0.4

deviation for measurement of 1 10 1 mol l 1

hemin was 2.8% (n= 10)

3.2. The ECL of lucigenin catalyzed by hemin

Hemin has been used as mimetic enzyme to
catalyze the CL reaction between luminol and
H2O2 [7]. We also found that hemin would catalyze the ECL of luminol at a platinum electrode in
a strong alkaline solution, however, the catalyzed
ECL emittance of luminol was much weaker than
CL emittance of luminol. Therefore, it was not
possible to be used for immunoassay. The ECL of
lucigenin in aqueous alkaline solution saturated
with oxygen at a platinum electrode has been
investigated [13], and the result indicated that the
ECL of lucigenin is emmited at the peak potential
of 0.30 V, but it was similar to luminol, the
sensitivity of the lucigenin ECL system was much
lower than its CL system. Our experiments
showed that the sensitivity of the ECL of Lucigenin would be increased greatly in the presence
of trace H2O2, and some metal ions would catalyze this system. Thus, there is no doubt that this
system can be applied to determination of trace
metals in a similar way to luminol, but it can be
used only for determination of metals, as lucigenin does not has any group which can be conjugated to other compound. Therefore it cannot be
used as the label and further used for immunoassay. Fortunately, we found that hemin
would catalyze the ECL of lucigenin in a neutral
KCl solution at a platinum electrode in the presence of trace H2O2. It has been confirmed that
hemin can be used as the label to conjugate to
protein, such as human serum albumin (HAS),
and this catalyst label can be sensitively quantitated by catalytic activity in a manner similar to
enzyme immunoassay [7].

G. Nan Chen et al. / Talanta 50 (2000) 12751281

3.2.1. The ECL of lucigenin in the presence of

hemin
Haapakkas experiments showed that lucigenin
would give ECL at 0.3 V in alkaline solution at
the platinum electrode [13]. We found that addition of trace H2O2 would greatly increase the ECL
of lucigenin at platinum electrode. Unfortunately,
as stated above the highly sensitive detection of
lucigenin has no significance in analytical practice
as it lacks of conjugation group.
It is well known that lucigenin will react with
H2O2 to give CL in alkaline solution, but not in
neutral solution. Conversely, lucigenin would give
ECL at a platinum electrode in neutral solution,
especially in the presence of H2O2 and hemin.
Therefore the neutral solution was used for subsequent experiment to avoid producing CL. As the
determination was based on the catalytic activity
of hemin, so the conditions should be optimized
to minimize the non-catalytic ECL and maximize
the catalytic ECL.

Fig. 1. ECL intensity of lucigenin as a function of potential

(Lucigenin)=1.0 10 6 mol l 1; (KCI)= 0.1 mol l 1.

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KCl was used as the electrolyte in our experiment. The effect of KCl on the catalytic ECL has
been examined and the result showed that KCl
would not effect the catalytic ECL in the range of
0.020.2 mol l 1, 0.1 mol l 1 was used for the
subsequent experiment.
The H2O2 concentration had a marked influence on the catalytic ECL intensity. The experiment showed that H2O2 can enhance catalytic
ECL intensity greatly and the enhancement of
intensity increases linearly with concentration of
H2O2 in the range of 1 10 5 5 10 5 mol l 1.
Above this concentration the catalytic ECL intensity decreases with increasing H2O2 concentration.
This is due to the increasing of non-catalytic ECL
(background) in higher concentration of H2O2.
Therefore, 3 10 5 mol l 1 H2O2 was selected
for subsequent experiment.
The effect of lucigenin concentration has been
examined and the results showed that the catalytic
ECL intensity would be increased with the concentration of lucigenin, but the non-catalytic ECL
(background) was increased as well. It was found
that when the concentration of lucigenin was 1
10 9 mol l 1 the difference between catalytic and
non-catalytic ECL could be maximized, therefore,
1 10 9 mol l 1 lucigenin was used in the subsequent experiment.
Linear sweep, square, triangular and symmetric
double step pulse voltages were selected to examine the catalytic ECL behavior of lucigenin at the
platinum electrode in the presence of hemin. It
was found that the catalytic ECL was stronger
when linear sweep was performed. Fig. 1 shows
the ECL intensity observed during the electrolysis
of lucigenin in the presence of hemin at the platinum electrode as a function of potential under
the stated conditions. The curve shows the intensity maximum with peak potential at 0.40 V
versus AgAgCl. Unlike non-catalytic ECL of
lucigenin, the light life of catalytic ECL of lucigenin is shorter, therefore, the intensity was related to the scan rate. The experiment showed
that the lower scan rate lower scan rate was better
than higher, the best scan rate was in the range of
1030 mV s 1; 20 mV s 1 was used in the
subsequent experiments.

G. Nan Chen et al. / Talanta 50 (2000) 12751281

1280

Table 3
Optimum conditions for the ECL of lucigenin catalyzed by
hemin
Electrochemical parameters

Waveform
Scan rate

Linear sweep
20 mV s1

Solution conditions
(mol l1)
KCl
H2O2 3105
Lucigemn

0.1
3105
1109

Lu2 + + e Lu +:
H2O2 + e OH + OH

Here, P* is the light-emitting species, which, most

probably peroxides, decompose via an intermediate of the emitter, N-methylacrdone [14].
Under the above optimum condition, the linear
logarithmic calibration range cover several order
of magnitude of concentration of hemin (1.0
10 10 1.010 15 g ml 1). The calibration
curve of catalytic activity hemin for ECL is shown
in Fig. 3. The minimum detectable concentration
of hemin (defined as the concentration which
could be detected for a signal-to-noise ratio of 3)
was 1.010 15 g ml 1, and the relation standard deviation for measurement of 1.0 10 12 g
ml 1 hemin is 7.5% (n= 10).

Fig. 2. Cyclic voltammegram of lucigenin (Lucigenin) =1.0

10 4 mol l 1; (KCl) =0.1 mol l 1. a: Lucigenin +KCI; b:
KCl.

The optimum conditions for the ECL of lucigenin catalyzed by hemin are shown in Table 3.
Fig. 2 presents the cyclic voltammegram of
lucigenin under the stated conditions. An irreversible reduction process of lucigenin can be
observed from Fig. 2. The peak potential of lucigenin reduction wave is 0.40 V versus Ag
AgCl, which can match the peak potential versus
ECL intensity.
It has been confirmed that hydrogen peroxide
should be involved in the ECL reaction of lucigenin at a platinum electrode. The formation of a
reduction product of lucigenin, which is most
probably a radical, is necessary for the lucigenin
to produce ECL [13]. Based on the results obtained by cyclic voltammetry and the intensity
versus potential curves, the following general reaction scheme is proposed for the ECL reaction of
lucigenin at the platinum electrode in the presence
of H2O2 and hemin,

Fig. 3. Calibration curves. , Catalytic activity hemin for

ECL; , conjugated hemin for ECL.

G. Nan Chen et al. / Talanta 50 (2000) 12751281

1281

4. Conclusion

Fig. 4. Sephadex G-25 chromatogram of hemin-IgG. , Absorbance; , ECL intensity.

3.3. ECL catalytic acti6ity of the conjugated

hemin-IgG
IgG labeled with hemin was used to examine
the ECL catalytic activity of hemin after
conjugating to protein. The Sephadex G-25
chromatogram of the hemin-IgG is shown in Fig.
4. Each fraction (3 ml) was monitored at 280 nm
for its absorbance and ECL intensity. It can be
seen from Fig. 4 that fraction 15 shows the maximum absorbance and ECL intensity. The
sharp peak in the elusion pattern is ascribed
to the hemin-IgG conjugated. No noticeable
fraction of free hemin was obtained, as the free
hemin was eliminated by dialysis prior to chromatography. Fraction 15 was used for further
investigation because of its higher hemin protein
molar.
Conjugated hemin has been tested for ECL
catalytic activity. Under the above mentioned optimum conditions, ECL intensity was measured at
different concentration of conjugated hemin
against fixing lucigenin and hydrogen peroxide
concentration. The linear response range was
1.0 10 14 1.0 10 10 g ml 1. The calibration
curve of conjugated hemin for ECL is also shown
in Fig. 3. Conjugated hemin can be quantitated in
this concentration range by measuring ECL intensity under the above conditions. It is obvious that
hemin retains ECL catalytic activity when conjugated to protein.
.

Three conclusions can be drawn: (1) hemin

displays ECL behavior at a platinum electrode in
alkaline solution when a triangular pulse voltage
is applied; (2) hemin would catalyze the ECL
reaction of lucigenin at a platinum electrode in
neutral solution in the presence of H2O2; (3)
hemin retains ECL catalytic activity when conjugated to protein, which indicates that hemin is
possible to be used as the mimetic enzyme for
labeling of antigen and antibody and further used
for immunoassay.

Acknowledgements
This project was financially supported by the
Natural
Sciences
Foundation
of
China
(296750003) and the Natural Sciences Foundation
of Fujian Province, China.

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