Bioluminescence Detection of Superoxide Anion Using Aequorin

Article abstract of DOI:10.1021/acs.analchem.9b02293

Although the superoxide anion (O₂ ^-·) is generated during normal cellular respiration and has fundamental roles in a wide range of cellular processes, such as cell proliferation, migration, apoptosis, and homeostasis, its dysregulation is associated with a variety of diseases. Regarding these prominent roles in biological systems, the development of accurate methods for quantification of superoxide anion has attracted tremendous research attention. Here, we evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter protein of superoxide anion. The mechanism is based on the measurement of aequorin bioluminescence, where the lower the concentration of coelenterazine under the oxidation of superoxide anion, the lower the amount aequorin regeneration, leading to a decrease in bioluminescence. The bioluminescence intensity of aequorin was proportional to the concentration of superoxide anion in the range from 4 to 40 000 pM with a detection limit (S/N = 3) of 1.2 pM, which was 5000-fold lower than those of the chemiluminescence methods. The proposed method exhibited high sensitivity and has been successfully applied to the determination of superoxide anion in the plant cell samples. The results could suggest a photoprotein-based bioluminescence system as a highly sensitive, specific, and simple bioluminescent probe for in vitro detection of superoxide anion.

Full text of DOI:10.1021/acs.analchem.9b02293

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Article
Bioluminescence detection of superoxide anion using aequorin
Hossein Rahmani, Fahimeh Ghavamipour, and Reza H. Sajedi
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02293 • Publication Date (Web): 09 Sep 2019
Downloaded from pubs.acs.org on September 12, 2019
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Bioluminescence detection of superoxide anion using aequorin
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*
Hossein Rahmani , Fahimeh Ghavamipour and Reza H. Sajedi
Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University,
Tehran 14115-154, Iran
1These authors contributed equally to this work.
*Corresponding author: R. H. Sajedi, Department of Biochemistry, Faculty of Biological
Sciences, Tarbiat Modares University, Tehran 14115-154, Iran, Fax/Tel: +98 21 82884717
E-mail: Sajedi_r@modares.ac.ir
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Abstract
−•
Although the superoxide anion (O ) is generated during normal cellular respiration and has
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fundamental roles in a wide range of cellular processes such as cell proliferation, migration,
apoptosis and homeostasis, its dysregulation is associated with a variety of diseases. Regarding
these prominent roles in biological systems, the development of accurate methods for
quantification of superoxide anion has attracted tremendous research attentions. Here, we
evaluated aequorin, a calcium-dependent photoprotein, as a potential bioluminescent reporter
protein of superoxide anion. The mechanism is based on the measurement of aequorin
bioluminescence, where the lower the concentration of coelenterazine under the oxidation of
anion superoxide, the lower the aequorin regeneration, leading to a decrease in the
bioluminescence. The bioluminescence intensity of aequorin was proportional to the
concentration of superoxide anion in the range from 4 to 40000 pM with the detection limit (S/N
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3) of 1.2 pM which was 1000-fold lower than that of chemiluminescence methods. The
proposed method exhibited high sensitivity and has been successfully applied to the
determination of superoxide anion in the plant cell samples. The results could suggest
photoprotein-based bioluminescence system as a highly sensitive, specific and simple
bioluminescent probe for in vitro detection of superoxide anion.
Keywords: Aequorin; Photoprotein; Bioluminescent; Coelenterazine; Superoxide anion; Reactive
oxygen species (ROS)
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Introduction
In a biological context, reactive oxygen species (ROS), as natural by-products of the normal
metabolism of oxygen, have significant roles in cell signaling and subsequent a wide range of
cellular processes including cell proliferation, migration, apoptosis and homeostasis.1–4 However,
some environmental stresses (e.g., UV or heat exposure), lead to a significant increase in ROS
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level. This may cause serious damage to cellular structures and predispose cells to malignant
transformation.6–10 Taken together, this is known as oxidative stress which is thought to play a
significant role in the initiation and progression of neoplastic, cardiovascular, renal and
neurodegenerative diseases.11 Moreover, ROS production in plants is strongly influenced by
some harsh environmental conditions, including salinity stress, drought, nutrient deficiency,
metal toxicity, chilling, UV-B and ionizing radiation.12,13
Enzymatic electron transfer from a reductant to molecular oxygen leads to production of oxygen-
derived free radicals in the body. As the products of a one-electron reduction of oxygen,
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•
-•
superoxide anions (O ), are the precursors of most other ROS. The reaction of O with nitric
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oxide yields the highly aggressive oxidant peroxynitrite (ONOO ) which largely limits nitric
oxide bioavailability. Superoxide dismutases (SOD) gently catalyze the dismutation of O2-• to
hydrogen peroxide (H O ) in the body. In the presence of trace metals, specially copper and iron,
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H O and O can produce hydroxyl radicals (OH ) which excessively react with every biological
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molecule. In various pathological conditions, several enzyme complexes, such as xanthine and
NAD(P)H oxidases can be triggered in many cellular systems to generate large amounts of
superoxide.15,16 Due to the short half-life and high reactivity of ROS, direct in vivo detection of
these radicals is really challenging. However, some molecules have been previously reported that
17
react specifically with ROS and can be applied for tracing of them. Enzyme activity assays,
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fluorimetric, colorimetric, and luminescence-based assays are used to detect radicals.18
Luminometric assays, in which light emission is enhanced in the presence of the studied radicals
as inducer, are more sensitive methods and allow temporal resolution of the signal.19,20 One of
the main problems for detecting radicals is the lack of specific and sensitive methods to measure
the oxidative stresses in vivo and in vitro. Different assays are developed to evaluate the
generation of free radicals, However, each method shows significant limitations.11
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As the prosthetic group of coelenterate and ctenophore photoproteins (e.g. aequorin and
mnemiopsin), coelenterazine, could also act as a conventional luciferin in certain mysids,
decapod shrimps, copepods, squid and fish.21–23 Coelenterazine, an imidazopyrazinone molecule,
is sequestered in the hydrophobic cavity of aequorin in a 2-hydroperoxycoelenterazine state.24,25
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Calcium binding to three Ca -binding EF hand loops of aequorin causes a slight conformational
change, leading to oxidation of the hydroperoxycoelenterazine to an excited coelenteramide.
Relaxation of the excited coelenteramide proceeds radiatively, emitting the blue light at
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maximum wavelength of 470 nm. The bioluminescence activity of these photoproteins depends
on the presence of a tightly but non-covalently bound coelenterazine.26 Since calcium ion
directly contributes to the reaction at saturating concentration, the emission of light is
proportional to the amount of the photoprotein. Photoproteins can be detected down to attomole
levels with a range of linear response extending for several orders of magnitude.27,28
Recombinant apophotoproteins are converted into photoproteins by incubation with
coelenterazine without any folding problems. All these factors participated in the rapid
development of photoproteins as bioluminescent reporters.29–31 As a well-known bioluminescent
photoprotein, aequorin is extensively used for a variety of analytical and biological applications,
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including as an intracellular calcium index, gene expression studies, drug discovery, and as
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a very sensitive reporter in immunological and binding assays.35 Due to its negligible
bioluminescence background, aequorin offers detection at very low concentration and no
interference with biological fluids and matrices, resulting in very low detection limit. regarding
this feature together with its non-toxicity, aequorin offer an advantage over the fluorescent or
colorimetric alternatives in certain types of studies.32
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The small molecule coelenterazine is known to react with the superoxide anion and peroxynitrite
and generate a chemiluminescent signal.23,36–39 Like bioluminescence, chemiluminescence
generates its own emission and does not require excitation light. Accordingly, there is no
background fluorescence except in the case of emission. Previous studies have indicated that
chemiluminescence from coelenterazine can be used to measure physiological superoxide anion
produced in living animal cells and in the cultured cells as well as the neutrophilic oxidative
burst in vitro.23,40,41 Coelenterazine is the substrate for the Gaussia and Renilla luciferases and
often administered for bioluminescence imaging in vivo. Taken together, in vivo delivery of
coelenterazine is considered safe.42–44 However, due to the low chemiluminescence signal
intensity, coelenterazine is not sensitive enough to detect low concentrations of superoxide
anion. To overcome this limitation, a relatively high concentration of coelenterazine should be
used. Here, we tried to improve the sensitivity of the method for measurement of superoxide
anion using photoprotein-based bioluminescence system. As the most convenient method of
-•
generating O , xanthine/xanthine oxidase system was applied here to produce superoxide anions
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which convert coelenterazine to coelenteramide, followed by measuring the changes in effective
concentration of coelenterazine by aequorin. More precisely speaking, coelenteramide, despite
being capable of binding to apoaequorin, cannot produce bioluminescence. Consequently, the
sensitivity of the assay can be increased by trapping the superoxide anion with coelenterazine
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and then measuring this reduction by aequorin. The higher the superoxide anion produces in the
reaction mixture, the lower the effective concentration of coelenterazine, leading to decrease in
the bioluminescence of aequorin (scheme 1). This feature of the proposed method has also
solved one of the most common problems in the measurement of superoxide anion which is very
short half-life and conversion to other forms of ROS.
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Scheme 1. Schematic illustration of superoxide anion assay using aequorin-based
bioluminescence system. Considering that coelenterazine concentrations are important in the
number of regenerated aequorin, any changes in the effective concentration of coelenterazine
leads to change in the bioluminescence intensity.
Materials and Methods
Chemicals and reagents
Xanthine, xanthine oxidase and superoxide dismutase (SOD) were acquired from Sigma (St.
Louis, MO, USA). The cp-coelenterazine was obtained from Resem BV (The Netherlands).
Ampicillin and isopropyl-D-thiogalactopyranoside (IPTG) were purchased from Bio Basic
Incorporation (Markham, Ontario, Canada). The Ni-NTA agarose was provided by Qiagen
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(
Qiagen, Hilden, Germany). All the other chemicals were obtained from Merck (Darmstadt,
Germany).
Expression and purification of apoaequorin
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The recombinant pET21a containing apoaequorin gene was transformed into Escherichia coli
BL21 (DE3) expression host. Following induction of His-tagged protein expression by adding
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mM IPTG for 4 h at 20 ˚C, cells were harvested by centrifugation and lysed by sonication.
Afterwards, the recombinant protein was purified by Ni-NTA agarose column described
previously.45
Preparation of semi-synthetic aequorin
To generate semi-synthetic aequorin from recombinant apoaequorin, the purified apoaequorin
was diluted with 50 mM Tris buffer (pH 7.6) containing 5 mM EDTA (buffer 1). This solution
was mixed with a given volume of cp-coelenterazine at a final concentration of 0.1 μM. The
regenerated mixture was briefly mixed and then incubated at 4 ˚C for 16 h.
Bioluminescence measurement
Bioluminescence emission intensities were recorded using Sirius L Tube Luminometer (Titertek-
Berthold, Germany) and the peak emission intensity was plotted. 100 μL of semi-synthetic
aequorin in 50 mM Tris buffer with pH 7.6 (buffer 2) at a concentration of 0.1 μM was pipetted
into a 1.5 ml microtube. The luminescence intensity was measured after injecting 100 μL of
buffer containing 50 mM Tris and 100 mM CaCl (buffer 3) for 5 sec with 0.05 sec time interval.
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Production and measurement of superoxide anion
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Xanthine/xanthine oxidase system was used to produce superoxide anion. Reaction mixtures
contained 0.1-0.5 U/ml of xanthine oxidase and 0.0006-600 µM of xanthine in 50 mM Tris
buffer (pH 7.0). The production of uric acid was measured as a function of time by monitoring
the increase in absorption at 290 nm using a UV S-2100 spectrophotometer. The concentration of
superoxide anion was determined according to the stoichiometry. To prepare various
concentrations of superoxide anion, different concentrations of xanthine (up to 150 µM and 0.6
mM for spectroscopic and chemiluminescence methods, respectively) were used. After the
addition of 0.5 U/ml xanthine oxidase, the concentrations of enzyme and substrate were
optimized to complete the reaction after 10 minutes.
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Chemiluminescence detection of superoxide anion using coelenterazine
Chemiluminescence detection of superoxide anion was carried out according to the method of
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Saleh and Plieth. 50 µM coelenterazine was added to the reaction mixture containing different
concentrations of xanthine (0.6 nM to 0.6 mM) and 7 mU/ml xanthine oxidase, followed by
measuring the bioluminescence intensity over 5 min.
Bioluminescence detection of superoxide anion using aequorin
In this method, the concentrations of xanthine and xanthine oxidase were the same as mentioned
in the chemiluminescence method. Coelenterazine was added at a final concentration of 0.1 µM
to the reaction mixture. After completing the enzyme reaction in 10 min, apoaequorin was added
to the reaction mixture at a final concentration of 0.1 µM and incubated at 4 °C for 16 h. The
bioluminescence intensity of aequorin was measured by injecting 100 μl of buffer 3.
Measurement of superoxide anion in real sample
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The applicability of the method for evaluating the concentration of superoxide anions in real
samples was assessed using Nicotiana tabacum cell. A magnetic field of 0.2 Tesla was used to
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induce the production of superoxide anion. 200 mg of plant cell was ground into fine powder
with liquid nitrogen. Further grinding was done in buffer 1. Following centrifugation of the
homogenates (14000 g, 15 min at 4 °C), 100 μl of the supernatant was mixed with coelenterazine
at final concentration of 0.1 µM. Apoaequorin was added to the reaction mixture after 10 min,
and the concentration of superoxide anion was then evaluated according to the
chemiluminescence and bioluminescence method described above. Also, as control, 25 unit/mL
of SOD were added to the supernatant of treated sample and after 20 min, the coelenterazine was
added to the mixture and the assay was performed as before.
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Anion superoxide detection in spiked samples
To verify the accuracy and precision of the method, a certain concentration of anion superoxide
(at a final concentration of 1 nM) produced by xanthine/xanthine oxidase system (3 nM xanthine
and 0.5 unit/mL of xanthine oxidase) was spiked into the supernatant of untreated plant cell
specimen in three replicates. The samples were analyzed following the assay procedure
described above and the average recovery and variation coefficient value were calculated.
Interference studies
Different concentration of hydrogen peroxide (1 nM to 1 mM) was added to the reaction mixture.
The concentration of superoxide anion was measured according to the method described above.
Effect of SOD on chemiluminescence and bioluminescence-based assay of superoxide anion
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5 - 2.5 mU/ml of SOD was added to the reaction mixture, and all other assay conditions were
the same as mentioned above.
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Statistical analysis
The statistical analysis and curve fitting were performed using GraphPad Prism software (San
Diego, CA). To analyze dose-response curves, a three parameters logistic equation (sigmoid)
was used (Y=Bottom + (Top-Bottom)/(1+10 ((X-LogIC50))), where X and Y are the logarithm of
concentration and response, respectively. Data are presented as the mean ± standard deviation
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(SD). Reproducibility of the data presented in this paper was confirmed by repeating the
experiments at least three times.
Results and discussion
Optimization of superoxide anion production
In this study, the xanthine/xanthine oxidase system was used as the source of superoxide anion
radicals (Figure 1A). The rate of the formation of uric acid from xanthine can be evaluated
photometrically on the basis of UV absorbance at 290 nm.47 Due to the mechanism of the
reaction and the type of electron transfer, for every six uric acid molecules, two molecules of
superoxide anion are produced, which is considered in the calculation of the superoxide anion
concentration.48 The production of superoxide anion was optimized considering to various
parameters, including the reaction time, substrate and enzyme concentration to gain the
maximum sensitivity of the bioluminescence system. Accordingly, the reaction was followed at
150 µM substrates and 0.1-0.5 U/ml enzymes in 30 min (Figure 1B). The results demonstrated
that by using 150 µM xanthine and 0.5 U/ml xanthine oxidase, the reaction was completed in 10
min.
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using xanthine as substrate. Oxidation of xanthine, to uric acid (UA) was catalyzed by xanthine
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oxidase, leading to formation of superoxide (O ) radical. Xanthine oxidase utilizes xanthine and
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O as substrate and produces uric acid and superoxide (O ). The progress curve of the xanthine
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oxidase reaction in the presence of 150 µM xanthine and 0.1 U/ml xanthine oxidase by
measuring the absorption of uric acid.
Superoxide anion assay by chemiluminescence method
As an alternative method for the above mentioned absorption assay and as a more sensitive
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method, chemiluminescence can be used for measuring superoxide anion. Various parameters
including reaction time, substrate and enzyme concentration were also investigated and
optimized to perform the chemiluminescence method. As previously, the results show that when
the superoxide anion reaches its highest level, the chemiluminescence of coelenterazine increases
to maximum intensity (Figure 2A).
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To determine the optimal concentration of coelenterazine and to investigate the linear
relationship between the concentrations of superoxide anion and chemiluminescence intensity,
different concentrations (25, 10, 5 and 1 µM) of coelenterazine were used. The results show that
chemiluminescence intensity increases with increasing the concentration of coelenterazine,
however, the concentration-dependent relationship does not differ significantly (Figure 2B).
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Finally, the lowest concentration of coelenterazine (1 µM), which showed the lowest EC (208
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nM), was used as the optimal concentration for subsequent experiments. It should be pointed out
that, the signal-to-noise ratio increases by reducing the concentration of coelenterazine, which
decreases the quality of the measurement.
As depicted in Figure 2C, an obvious linear relationship has been observed between the
concentration of superoxide anion (20-20000 nM) and the chemiluminescence intensity of
coelenterazine (LOD 6 nM). As previously reported by Saleh et al, the activity of superoxidase
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was successfully measured by this method. Since the signal intensity in this method depends on
the concentration of coelenterazine, the relatively high concentrations of coelenterazine and high
sensitive devices should be used for the assay, thereby limiting the use of this approach. In this
study, to overcome the limitations of this method as well as to increase sensitivity, superoxide
anion was measured using aequorin-based bioluminescence system.
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Figure 2. (A) Chemiluminescence kinetic curve of coelenterazine (25 μM) in the presence of
different concentrations of superoxide anion. (B) A dose-response curve of coelenterazine
chemiluminescence against different concentrations of superoxide anion produced by the
xanthine/xanthine oxidase system. Chemiluminescence values of all samples were expressed as
the percentage of the maximum chemiluminescence intensity (CTZ: coelenterazine). (C) The
standard curve represents a linear relationship between RLU/S and different concentrations of
superoxide anion for the optimal concentration of coelenterazine (1 μM).
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Superoxide anion assay using photoproteins-based bioluminescence system
Given that the basis of the proposed method is the dependency of aequorin bioluminescence on
coelenterazine concentration and by virtue of the applicability and suitability of coelenterazine
for detection of superoxide anion, we next aimed to determine if aequorin-based
bioluminescence system could detect changes in superoxide anion concentrations. Different
amounts of superoxide anion were added to coelenterazine, followed by the addition of
apoaequorin to form a holo-aequorine. The bioluminescence reaction could be triggered by the
addition of calcium to the sample. Obviously, the more superoxide anion is produced in the
reaction medium, the lower effective concentration of coelenterazine, leading to a decrease in the
aequorin bioluminescence.
An inhibition curve for the superoxide anion was constructed by plotting bioluminescence
inhibition of aequorin against the target concentration (Figure 3A). The IC50 of the
bioluminescence was achieved at 430 pM; the limit of detection (LOD), which was defined as
the inhibition concentration of 10% (IC ), was 1.2 pM. The dynamic range was determined as
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the analyte concentration generating between 20% and 80% of maximal inhibition. As shown in
Figure 4B, the method exhibits excellent linearity (a correlation coefficient of 0.9834) within the
range of 4–40000 pM with a calibration curve of Y = -61845X-453148. The sensitivity of the
proposed bioluminescence method is approximately 1000-fold higher than those of the
previously reported chemiluminescence-based method.
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Figure 3. (A) Effect of different concentrations of superoxide anion on the bioluminescence
intensity of aequorin. (B) The method is able to detect the superoxide anion in a linear range
from 4 to 40000 pM.
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The effect of SOD on chemiluminescence and bioluminescence-based assay of superoxide
anion
Since the SOD specifically converts the superoxide anion to hydrogen peroxide, it can be used to
demonstrate the specificity of the method after the production of superoxide anion by the
xanthine/xanthine oxidase system in both chemiluminescence and bioluminescence methods. In
the bioluminescence method, SOD suppresses its reaction with coelenterazine, converting it to
coelenteramide and reducing the photoprotein emission by consuming anion superoxide.
Thereby the effect of SOD on bioluminescence and chemiluminescence-based assay of
superoxide anion was investigated to confirm that the changes of aequorin bioluminescence and
coelenterazine chemiluminescence intensity is due to the increase in superoxide anion
concentration not the other species. In chemiluminescence-based assay, after initiating the
enzymatic reaction of superoxide anion production and reaching the maximum of the
coelenterazine chemiluminescence intensity, different concentration of SOD was injected into
the reaction mixture. As shown in Figure 4, immediately after the injection of SOD, a significant
drop in chemiluminescence of coelenterazine occurs, which is proportional to the amount of
enzyme concentration. In bioluminescence-based assay when coelenterazine was mixed with a
reaction mixture containing xanthine and xanthine oxidase, the addition of aequorin led to a
bioluminescent reaction. Since the consumption of superoxide anion by SOD prevents its effect
on coelenterazine, the bioluminescent intensity of aequorin increases. Figure 5B shows the effect
of different concentrations of SOD (25-0.0025 U/mL) on the reaction mixture of xanthine and
xanthine oxidase. So the sensitivity of methods to SOD suggests that the changes in luminescent
intensity are due to the increase in the superoxide anion concentration.
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Figure 4. The effect of different amounts of SOD on (A) chemiluminescence and (B)
bioluminescence-based assay of superoxide anion (CTZ: coelenterazine).
Interference study
Since the biological environments contain many other types of ROS in addition to superoxide
anion, the specificity of the detection methods to measure each of these radical species has a
great deal of importance. Here, the interference of H O , as the most common type of ROS, was
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investigated in the bioluminescence detection of superoxide anion. The results showed that H O
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had no effect on aequorin bioluminescence intensity up to the concentration of 100 µM. Taking
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into account its usual range in biological samples, H O could not interfere with the detection
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method (Figure 5). In summary, the method exhibited favorable selectivity for the determination
of superoxide anion.
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Figure 5. Effect of different concentrations of H O on bioluminescence intensity of aequorin.
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Determination of superoxide anion in real sample
To better understand the efficiency and feasibility of our approach versus chemiluminescence
methods, incurred and negative plant cells of Nicotiana tabacum were analyzed using both the
monitoring platforms. The magnetic field was applied to induce superoxide anion production in
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the cells as previous report . Each sample was analyzed in triplicate and levels were determined
using the calibration curve (Figure 3B). Initially, superoxide anion assay was performed using
different concentrations of coelenterazine by the chemiluminescence method, which showed that
the treated samples had higher chemiluminescence intensity than control due to the higher level
of superoxide anion in these samples. As shown in Figure 6A, the higher the concentration of
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coelenterazine, the greater the chemiluminescence intensity. However due to the lower
sensitivity (LOD = 6 nM) of the chemiluminescence method, it is not possible to quantify this
level of anion superoxide produced in the treated sample by this method because this value is not
in the linear range of the chemiluminescence method and the level of superoxide anion in the
treated and untreated groups can only be evaluated for qualitative comparison.
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Subsequently, for comparative purpose, the amount of superoxide anion in samples was
evaluated using aequorin-based bioluminescence method (Figure 6B). The concentration of
superoxide anion in untreated and treated samples was determined 0.1 and 1.17 nM respectively
by bioluminescence method. As shown in Figure 6B, the difference between treated and
controlled samples can only be detected at high concentrations of coelenterazine in
chemiluminescence method and the signal intensity is nearly 10 times lower than that of the
proposed method. These results confirm the usefulness of aequorin as a very specific and
sensitive bioluminescence probe for superoxide anion and this is one of the advantages of our
method over the chemiluminescence methods that can detect this low level of anion superoxide.
Meanwhile, in both luminescence and bioluminescence methods the SOD-treated group was
investigated to determine the contribution of superoxide anion to the produced signal.
Interestingly, it has been shown that the major contribution of ROS produced in cells was due to
superoxide anion.
Accuracy was next examined using the recovery of spiked sample. Parallel samples were
prepared and determined by the method. Resulting levels in the spiked samples were deducted
from the control sample and then quantified using the calibration curve. The calculated recovery
value and CV are 90.5 ± 3.9% and 7.1%, respectively. These results revealed that the precision
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of the aequorin-based bioluminescence method for superoxide anion quantification was
acceptable.
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Figure 6. Superoxide anion determination by coelenterazine-based chemiluminescence assay
using 10, 1 and 0.1 µM of coelenterazine (A) and aequorin-based bioluminescence method (B) in
plant cells of Nicotiana tabacum under magnetic field stress versus untreated and SOD treated
samples. The concentration of 0.1 μM coelenterazine was used for both methods.
Conclusion
Here, the dependency of aequorin bioluminescence to coelenterazine, was used to measure
superoxide anion, as one of the common species of ROS. These results indicate the usefulness of
aequorin as a bioluminescent indicator for assaying superoxide anion. The proposed method has
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advantages like requiring neither high concentrations of coelenterazine nor a large sample
volume. Furthermore, it seems to be specific to superoxide anion, as may be concluded from the
influence by SOD. The plotted parameters are linearly correlated when the concentration of
superoxide anion is between 4-40000 pM with LOD of 1.2 pM. Sensors for biological molecules
are most useful when they can be applied in biological environments. The photoproteins-based
bioluminescence system provides that opportunity by measuring the superoxide anion in plant
cells in which superoxide anion was produced through a magnetic field. Comparison between the
linear range of bioluminescence and chemiluminescence methods shows that the proposed
method is more sensitive for determining the concentration of superoxide anion. Also, due to the
use of lower concentrations of coelenterazine, our method seems to be more cost-effective than
the chemiluminescence method so that the production signals are increased by aequorin.
Collectively, these results establish a new method allowing simple, selective and quantitative
detection of superoxide anion which can easily be applicable for real samples.
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Acknowledgments
The authors express their gratitude to Tarbiat Modares University for financial support during
the course of this project under the grant number IG-39708.
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