Retraction for Analyst: Production of Monoclonal Antibody against Mercury (II) Ion and “Turn-on” Chemiluminescence for Mimic Sandwich ELISA Detection of Hg2+ Ions Sheng Cai, Yuzhen Wang, Anping Deng and Jianzhong Lu Analyst, 2012, DOI: 10.1039/C2AN35303B. Retraction published 19th June 2012
We the authors Sheng Cai, Yuzhen Wang, Anping Deng and Jianzhong Lu, hereby wholly retract this Analyst article. This article contains antibody preparation results of research which had been submitted for publication in Analytical and Bioanalytical Chemistry, at an earlier date. The overlap of results was not intentional and this Analyst article is being retracted by the authors in order to maintain the accuracy of the scientific record. Signed S. Cai, J. Lu, Fudan University and Y. Wang, A. Deng, Soochow University, China, 19th June 2012. This retraction is endorsed by May Copsey, Editor. Retraction published 19th June 2012.
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Production of Monoclonal Antibody against Mercury (II) Ion and
“Turn-on” Chemiluminescence for Mimic Sandwich ELISA Detection of
Hg2+
Ions
Sheng Caia, Yuzhen Wang
b, Deng Anping*
b, and Jianzhong Lu*
a 5
Received (in XXX, XXX)1st January 2007, Accepted 1st January 2007
First published on the web 1st January 2007
DOI: 10.1039/b000000x
Mercury (Hg2+) ion is one of the most toxic heavy metals present in the environment. Driven by the
need to detect trace amounts of Hg2+ ions in environmental samples, this article demonstrates for the 10
first time that a new monoclonal antibody against Hg2+ ions is produced and then a mimic sandwich
chemiluminescence (CL) method is employed for rapid, easy and reliable detection of Hg2+ ions in
aqueous solution. Briefly, a new ligand 6-mercaptonicotinic acid (MNA) is coupled with both
methylmercury chloride (CH3ClHg) and carrier protein and then the thus formed CH3Hg-MNA-
bovine serum albumin conjugate is used as an immunogen. After immunizing BALB/c mice, spleen 15
cells of immunized mice are fused with myeloma cells and the monoclonal antibody (mAb) against
Hg2+ ions is produced by hybridoma technique. The immobilized mAb is then employed to capture
Hg2+ ion in the sample and gold nanoparticles (Au NPs) are thus formed due to the accelerated
catalysis of the mAb captured Hg2+ ions on the HAuCl4/NH2OH reaction. The Au NPs triggers the
AgNO3/luminol reaction to emit strong CL. Similar to a sandwich ELISA, herein the Au NPs acts like 20
a mimic detection antibody and CL intensity increases with the increase in the concentration of Hg2+
ion. This mimic sandwich CL method has several advantages including high sensitivity (0.008 ppb)
and selectivity over alkali, alkaline earth (Li+, Na+, Ca2+), and transition heavy metal ions (Pb2+,
Mn2+, Fe3+, Cu2+, Ni2+, Zn2+, Cd2+, Ba2+, Zr2+, Sr2+, Ag+), which makes the technology very attractive
for Hg2+ ions monitoring in environment, water, and food samples. 25
Introduction
Environmental pollution by heavy metals is a growing problem
worldwide, especially in developing countries. Mercury (Hg2+) is
one of the most toxic elements that can accumulate easily in
human bodies from the hydrosphere and aquatic food chain,1, 2 30
which has been monitored using several traditional detection
techniques, such as atomic absorption spectrometry,3, 4 atomic
fluorescence spectrometry5 and inductively coupled plasma mass
spectrometry.6, 7 While these methods are sensitive and accurate,
they are time-consuming and require sophisticated equipment, 35
generally in a laboratory setting. Consequently, there is a need for
Hg2+ detection methods with suitable selectivity and sensitivity
and low costs. Much effort has been devoted towards the design
of sensing systems for Hg2+ ions, including sensors based on
fluorophores,8-12 conjugated polymers,13 DNAzymes,14 gold 40
nanoparticles,15-17 proteins18, 19 and thymine–Hg2+–thymine base
pairs.20, 21
Immunoassays offer an alternative approach, and they have
significant advantages over the traditional instrument-intensive
methods of metal analysis. They are remarkably quick, simple 45
and portable for use in the field, require minimum sample
pretreatment, and have high throughput. One of the most useful
of the immunoassays is the sandwich ELISA. The sandwich
ELISA requires two antibodies that bind to epitopes that do not
overlap on the antigen. When a matching pair of antibodies is not 50
available for the target, another option is competitive ELISA.
However, for competitive ELISA, higher sample antigen
concentrations lead to a weaker signal.
Several monoclonal antibodies against metals, including
cadmium, lead, chromium, uranium and Hg2+,22-28 have been 55
produced using ligands such as glutathione, ethylenediaminetetra-
acetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA),
trans-cyclohexyldiethylenetriaminepentaacetic acid (CHXDTPA) 29 and 1,10-phenanthroline-2,9-dicarboxylic acid (DCP) (Figure
1). However, because the metal ion is typically enclosed by the 60
ligands, the antibodies produced are likely to be specific to the
metal-ligand complex rather than the metal ions. Consequently,
samples must be pre-treated with these ligands to form metal-
ligand complexes before analysis. Because of the small size of
the metal ions, only one antibody is produced and thus a 65
competitive ELISA needs to be used for quantification of the
metal ions, which means the signal will decrease as the amount of
metal ions is increased. In the present study, based on the strong
binding of Hg2+ with mercapto groups, 6-mercaptonicotinic acid
(MNA, Figure 1) was selected for complex formation with Hg2+ 70
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ions. With a carrier protein (BSA/OVA) this was used to produce
a sensitive and specific monoclonal antibody (mAb) against the
Hg2+ ions, not the Hg-ligand complex.
Scheme 1. Schematic representation of “turn-on” chemiluminescence for 5
mimic sandwich detection of mercury (II) ion.
Our interest in Hg2+-sensing issues stems from the discovery of
the HAuCl4/NH2OH reaction, which could be accelerated by the
Hg2+ ions.30, 31 Motivated by this observation, we report herein 10
that, the HAuCl4/NH2OH reaction can also be accelerated by the
antibody captured Hg2+ ions, even there is no gold nanoparticles
(Au NPs) in the solution. The solution turned red when Au NPs
formed on the backbone of the antibody in the HAuCl4/NH2OH
reaction. The thus formed Au NPs catalyzed the AgNO3/luminol 15
reaction to emit strong chemiluminescence (CL).32 The CL
intensity increased with the increase in the concentration of Hg2+
ions. Thus, similar to a sandwich ELISA, the Au NPs acted like a
mimic detection antibody and high concentrations of Hg2+ ions in
the sample gave strong CL signals. The principles of this mimic 20
sandwich ELISA CL detection of Hg2+ ions are shown in Scheme
1.
Experimental
Materials. 25
All chemicals were of analytical reagent grade and were used
as received. Water was purified using a Millipore Milli-XQ
system (Bedford, MA). Carboxyl-modified Nunc F96
MircroWell plates were obtained from Nunc Incorporated.
NH2OH, HAuCl4, Na2HPO4, NaH2PO4 and NaCl were 30
purchased from Sinopharm Chemical Reagent Co. Ltd.
Methylmercury chloride (CH3ClHg), 6-mercaptonicotinic acid
(MNA), mercuric chloride (HgCl2), 3,3’,5,5’-
tetramethylbenzidine (TMB), bovine serum albumin (BSA),
ovalbumin (OVA), dimethyl sulfoxide (DMSO), 35
dimethylformamide (DMF), N,N’-dicyclohexylcarbodiimide
(DCC), N-hydroxysuccinimide (NHS), Freund’s complete and
incomplete adjuvants, horseradish peroxidase labeled goat
anti-mouse IgG conjugate (HRP-GaMIgG), hypoxanthine
aminopterin thymidine (HAT), hypoxanthine thymidine (HT), 40
polyethylene glycol (PEG4000) were purchased from Sigma
Chemical Co. (St Luis, Mo. USA). RPMI 1640 was bought
from GibcoBri (Paisley, Scotland). Cell medium and fetal calf
serum was from Minhai (Lanzhou, China). Mouse SP2/0
myeloma cell was bought from the Cell Bank of Chinese 45
Science Academy (Shanghai, China). BALB/C mice were
purchased from Experimental Animal Center of Sichuan
University (Chengdu, China).
Figure 1. The structures of reported ligands for the immunoassay of 50
heavy metals, and 6-mercaptonicotinic acid (MNA) used as a ligand
to couple with both CH3ClHg and the carrier protein.
Apparatus.
CL measurement was carried out using a PC-controlled 55
Fluoroskan Ascent FL (Thermo Electron Corporation).
Absorbance was determined by a HITACHI U-2900
Spectrophotometer. CO2 incubator (HF 151 UV) was from
Heal Fore Development Ltd. (Shanghai, China). ELISA reader
(Sunrise Remote/Touch Screen) and microtiter plate washer 60
(M12/2R) were bought from Columbus plus (Tecan, Grödig,
Austria).
Buffers and solutions.
(1) Coating buffer: 0.05 mol/L carbonate buffer, pH 9.8; (2)
coating antigen stock solution: 1 mg/mL of coating antigen 65
prepared with coating buffer; (3) assay buffer: 0.01 mol/L
phosphate-buffered saline (PBS) pH 7.4, containing 145
mmol/L NaCl; (4) washing buffer (PBST): assay buffer with
0.1% (v/v) of Tween-20; (5) blocking solution: 1% of casein
in assay buffer; (6) acetate buffer: 100 mmol/L sodium acetate 70
acid buffer, pH 5.7; (7) substrate solution (TMB+H2O2): 200
µL of 10 mg/mL TMB dissolved in DMSO, 20 µL of 5% H2O2
and 1 mL of acetate buffer were added to 20 mL of pure water;
(8) stop solution: sulfuric acid (5 %); (9) Hg2+ ions stock
solution (1 mg/mL): 6.77mg HgCl2 was dissloved in 2% (v/v) 75
HNO3 and kept at 4 ℃;(10) Hg2+ ions standard solutions at
the concentrations of 0, 0.1, 0.3, 1.0, 3.0, 10, 30 and 100
ng/mL were prepared by diluting the stock solution with
ultrapure water.
Synthesis of MNA-Protein Conjugates. 80
The MNA was conjugated to BSA and OVA by the DCC/NHS
ester method (9). Briefly, equimolar amounts (0.06 mmol ) of
MNA, NHS, and DCC were dissolved in 200 µL of DMF and
the reaction was incubated overnight with stirring. After
centrifuging solution at 12,000 rpm for 10 min, the 85
supernatant was added slowly to 40 mg BSA or OVA in 3 mL
of 0.13 mol/L NaHCO3 under stirring. After reaction for 4 h
and centrifugation, the supernatant was dialyzed in 0.01 mol/L
PBS for 4 days.
Preparation of immunogen and coating antigen. 90
CH3ClHg (0.05 mmol) was dissolved in 200 µL of methanol
containing 10 % of NaOH (v/v). The solution of CH3ClHg
was added dropwise to MNA-BSA/OVA while stirring and
the reaction was incubated overnight. The next day, the
solution was dialyzed in 0.01 mol/L (NH4)2CO3 for 4 days 95
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with several changes of the dialyzing buffer solution. Finally,
the CH3Hg-MNA-protein conjugates were lyophilized for use.
Production of monoclonal antibody.
Two female BALB/C mice were immunized with 100 µg of
CH3Hg-MNA-BSA subcutaneously emulsified with an equal 5
volume of Freund’s complete adjuvant. In the next two
sequential booster immunizations, 100 µg of immunogen
emulsified with the same volume of incomplete Freund’s
adjuvant was given to each mouse in the same way at 2-week
intervals after the initial immunization. The fourth injection 10
was given intraperitoneally without adjuvant before cell
fusion. Three days after the final booster injection, spleen
cells were fused with mouse SP2/0 myeloma cells using 50 %
polyethylene glycol 4000 which was used as fusion agent. The
fused cells (hybridomas) were distributed in 96 well culture 15
plates supplemented with hypoxanthine aminopterin
thiamidine (HAT) medium containing 20 % fetal calf serum
with peritoneal macrophages as feeder cells from young
BALB/C mice. The growth of hybridomas in the plates was
incubated at 37 ℃ with 5 % CO2. After incubated for about 2 20
weeks, positive clones were screened by indirect enzyme
linked-immunosorbent assay (ELISA) using CH3Hg-MNA-
OVA as coating antigen. In the screening step, MNA,
CH3ClHg, Hg2+ and CH3ClHg-MNA were respectively as
competitors. The hybridomas were subcloned for three times 25
using the limiting dilution method. Stable antibody-producing
clones were expended and cryopreserved in liquid nitrogen.
Ascitic were produced in mice by injecting hybridoma cells
intraperitoneally which were preinjected with 0.5 mL of liquid
paraffin 1 week ago. Antibodies were collected and subjected 30
to purification by ammonium sulfate precipitation. The
purified mAb was stored at −20 ℃ in the presence of 50 %
glycerol. The specificity of the produced mAb was
investigated by the cross-reactivity (CR) experiment using
indirect competitive ELISA where the CH3Hg-MNA-OVA 35
conjugate was used as coating antigen. Different chemicals
such as Cu2+, Cr3+, Sn2+, Ni2+, Mn2+, Pb2+, Zn2+, Cd2+, Fe3+,
Co2+, Mg2+ Ag+, MNA, CH3ClHg and CH3Hg-MNA were
selected for testing CR. The standard solutions of cross-
reacting metal ions and compounds were prepared in the 40
concentration range of 0.001–1000 ng/mL. CR was expressed
as percent IC50 values based on 100% response of Hg2+ ions,
e.g. CR (%) = [IC50 for Hg2+ ions]/[IC50 for competing
chemicals] × 100 %. IC50 is the concentration of Hg2+ ions or
competing chemicals that produce a 50 % inhibition of the 45
signal.
CL assay procedures on polystyrene microwells.
In a typical experiment, mAb was diluted to 0.5 µg per 100 µL
in coupling buffer (0.01 M Na2HPO4-NaH2PO4, 0.15M NaCl,
pH 7.4) and placed in a 96-well plate (100 µL per well). The 50
wells were washed three times with washing buffer (8 mM
Na2HPO4, 2 mM NaH2PO4, pH 7.4, 0.9% NaCl, 0.05% Tween
20) after incubating with gentle mixing for 1 h at 37 °C.
Different amounts of Hg2+ or nontarget ions in 100 µL of
PBSC (8 mM Na2HPO4, 2 mM NaH2PO4, pH 7.4, 0.9% NaCl) 55
were then added into each well. Following incubation for 1 h
with gentle mixing at 37 °C, the wells were washed three
times with washing buffer. Then 50 µL of 40 mM NH2OH and
0.5 mM HAuCl4 were added and the mixture was incubated at
25 °C for 20 min. The wells were washed three times with 60
washing buffer, 50 µL of 10 mM luminol (0.1 M NaOH) was
pipetted into the microwells. Finally, 50 µL of 0.5 mM
AgNO3 solution was injected into a Fluoroskan Ascent FL and
the CL signal detected. For the amplification assay, Au NPs
that assembled on the surface of the 96-well plate were 65
catalytically enlarged in the presence of 40 mM NH2OH and
0.5 mM HAuCl4 at 25 °C for 10 min. The wells were washed
three times with washing buffer, and then the CL signal was
detected as described above.
70
Results and Discussion
The ligand MNA was first linked to protein by the DCC/NHS
ester method, and then the MNA-protein conjugate was
coupled with CH3Hg-Cl to form CH3Hg-MNA-protein. Two
female BALB/C mice were immunized subcutaneously with 75
100 µg of CH3Hg-MNA-BSA, which was emulsified with an
equal volume of Freund’s complete adjuvant. The mice were
then injected subcutaneously at 2-week intervals with two
sequential booster immunizations, which contained 100 µg of
immunogen emulsified with the same volume of incomplete 80
Freund’s adjuvant. fter the third injection, antisera collected
from the two immunized mice displayed high affinity binding
with the coating antigen CH3Hg-MNA-OVA. Three days after
the final booster injection, the mice were sacrificed and their
spleens removed. The spleen cells from the two mice were 85
used for fusion experiments. After incubation for about 2
weeks, the supernatants from the hybridoma cells were
screened by indirect ELISA. The hybridomas, which were
positive to CH3Hg-MNA-OVA and negative to MNA-OVA,
were subcloned three times using the limiting dilution method. 90
Figure 2. Molecular structure of CH3Hg-MNA (1) and molecular
model of CH3Hg-MNA (2) (mercury, dark grey; hydrogen, white;
carbon, grey; sulfur, yellow; nitrogen, dark blue; oxygen, red).
95
The initial aim of this experiment was to obtain a
monoclonal antibody against CH3ClHg instead of Hg2+ ions.
However, the Hg2+ ions, instead of MNA, CH3ClHg and
CH3ClHg-MNA, displayed a strong inhibition in the indirect
ELISA when the supernatant from the hybridoma cells of the 100
positive clone was used as the antibody. CR values of the
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mAb with MNA, CH3ClHg and CH3ClHg-MNA were found to
be 0.72 %, 1.97 % and 1.16 %, respectively; no cross-
reactivity was with other ions such as Cu2+, Cr3+, Sn2+, Ni2+,
Mn2+, Pb2+, Zn2+, Cd2+, Fe3+, Co2+ and Mg2+, except lesser
than below 10 % CR value with Ag+. The CR values strongly 5
indicate that a hybridoma producing specific antibody against
Hg2+ ions was successfully screened out and the produced
mAb displays high specificity for Hg2+ ions. The structure of
CH3Hg-MNA (Figure 2) may have contributed to this result,
because the Hg2+ ion is almost completely exposed on the 10
MNA, which increases exposure of the Hg2+ ions to the
animal’s immune system.
Figure 3 displays the absorbance spectra for Au NP
formation catalyzed by either free or antibody captured Hg2+
in the HAuCl4/NH2OH reaction. The maximum absorption 15
wavelength was >600 nm for free Hg2+ ions, while the
maximum absorption wavelength was 550 nm for the antibody
captured Hg2+ ions (Figure 3). A blue shift was observed for
the formation of Au NPs by the antibody captured Hg2+ ions
in the HAuCl4/NH2OH reaction, which indicates that small Au 20
NPs formed. It is well known that metallic NPs are unstable
and have a tendency to aggregate. The use of antibody is very
significant to prevent the aggregation and maintain the
stability of Au NPs in aqueous solution. The transmission
electron microscope images show that the average diameter of 25
the Au NPs was 20 nm for the antibody captured Hg2+
catalyzed HAuCl4/NH2OH reaction whereas that the Au NPs
generated from the free Hg2+ catalyzed HAuCl4/NH2OH
reaction were 200 nm in diameter.
30
Figure 3. Absorbance of Au NPs and TEM images for the
HAuCl4/NH2OH reaction catalyzed by free Hg2+ (red spectrum, right
photo) and antibody captured Hg2+ (black spectrum, left photo), scale bar:
100 nm. Experimental conditions: mAb = 0.5 µg; Hg2+ = 200 ng/mL;
NH2OH and HAuCl4 concentrations were 40 and 0.5 mM, respectively. 35
The detection procedure was carried out as described in the Experimental
section.
Optimization of Reaction Parameters. Several parameters
were investigated systematically to establish optimal 40
conditions for the ultrasensitive mimic sandwich ELISA Hg2+
detection, including the amounts of mAb, HAuCl4, NH2OH,
AgNO3 and luminol, etc.
As shown in Figure S1, with the increase of the amount of
mAb, CL intensity was observed to increase over the range of 45
0-0.5 µg of mAb and then decreased slowly. It was postulated
that the decrease was due to steric and electrostatic
hindrances, arising from more tightly packed capture antibody
on the plate surface. Thus, 0.5 µg mAb was selected for
subsequent experiments. 50
The effects of the concentration of HAuCl4 and NH2OH
were subsequently examined and optimized. The CL signal
intensity increased with increasing concentration of HAuCl4
in the range 0.01–0.5 mM, and then decreased in the range
0.5–2 mM (Figure S2). The CL intensity increased in the 55
range 0.1–40 mM of NH2OH and then remained almost
constant (Figure S3). Thus, 0.5 and 40 mM of HAuCl4 and
NH2OH were selected as the amounts for use in further
studies.
In addition, the concentrations of luminol and AgNO3 also 60
affected the CL signal. Therefore, these parameters were also
examined and optimized. First, it was found that CL intensity
increased with increasing luminol concentration, reached a
maximum and then remained almost constant after a
concentration of 10 mM (Figure S4). For AgNO3, CL intensity 65
increased over the range of 0.03-0.5mM, and then decreased
(Figure S5). Hence, 10 mM luminol and 0.5 mM AgNO3 were
selected for subsequent experiments.
The incubation time of NH2OH and HAuCl4 played an
important role in the detection. In the first 16 min, CL 70
intensity increased as the blank signal was low. With the time
going, blank signal increased fast and the CL intensity and CL
ratio decreased. Then, we chose 20 min as the incubation time
(Figure 4).
Figure 4. CL intensity (■) and CL ratio (◆) vs the incubation time. 75
Experimental conditions: mAb was 0.5 µg; Hg2+ was 200 ng/mL;
NH2OH, HAuCl4, luminol and AgNO3 were 40, 0.5, 10 and 0.5 mM,
respectively. The detection procedure was carried out as described in the
Experimental section.
80
Analytical Performance of Hg2+ Detection. Under optimal
conditions, this assay was challenged with an increasing
amount of Hg2+, which resulted in a dynamic increase in the
CL intensity. Figure 5 shows the increase in CL as a function
of the Hg2+ amount. This relationship was linear from 0.1 to 85
1000 ng/mL, and is represented by LgI = 0.3691LgC+3.3254
(R2=0.9819), where I is the CL intensity and C is the
concentration of Hg2+. The limit of detection (3σ, n=5) was
0.4 nM (0.08 ppb), which is comparable with most previous
assay techniques (Table 1). However, this technique has fewer 90
steps and a shorter assay time than the other techniques. After
amplifying the HAuCl4/NH2OH reaction, the detection limit
improved (0.04 nM, 0.008 ppb). Note that the use of
antibodies for Hg capture greatly improved the sensitivity and
the limit of detection was 250 times more sensitive than 95
previous detection of free Hg2+ ions in the solution with
HAuCl4/NH2OH reaction.30 We reasoned that this
improvement was caused by two main factors, i.e. Hg
preconcentration and the anti-aggregation and protection of
the formed Au NPs by the use of antibodies, and thus the 100
actual exposed surface of Au NPs was increased, leading to an
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improved sensitivity.33 Therefore, the use of antibodies for Hg
capture will be more suitable for the detection of Hg2+ ions in
environment, water, and food samples. To the best of our
knowledge, this is the lowest detection limit ever reported for
a Hg2+ ions sensing system without signal and PCR 5
amplification enzymes.water, and food samples.
Figure 5. Log-log calibration data for Hg2+ ions without (◆) and with
(■) enlargement. Experimental conditions: mAb = 0.5 µg; NH2OH,
HAuCl4, luminol and AgNO3 concentrations were 40, 0.5, 10 and 0.5 10
mM, respectively; and the incubation time was 20 min. The detection
procedure was carried out as described in the Experimental section.
Detection Specificity. The selectivity of the method was
investigated by testing the response of the assay to other metal 15
ions, including Cd2+, Ba2+, Ca2+, Pb2+, Ni2+, Cu2+, Zn2+, Mn2+,
Fe3+, Li+, Zr2+, Sn2+ and Ag+ at a concentration of 100 µM
under the same experimental conditions as for Hg2+.
Remarkably, very little increase in CL was observed with
other metal ions, even when they were at a 100-fold higher 20
concentration than Hg2+ (Figure 6). These results demonstrate
this method has excellent selectivity for Hg2+ over alkali,
alkaline earth, and heavy transition-metal ions. In
addition, organic mercury such as methylmercury also did not
interfere with the detection of Hg2+ ions. The specific 25
detection for Hg2+ ions can be attributed to both the high
affinity and specificity of mAb and the highly selective Hg2+
catalyzed HAuCl4/NH2OH reduction reaction.
Figure 6. Selectivity of the analysis of Hg2+ ions by the method depicted 30
in Scheme 1 with the following metal ions: 1, Hg2+; 2, Cd2+; 3, Ba2+; 4,
Ca2+; 5, Pb2+; 6, Ni2+; 7, Cu2+; 8, Zn2+; 9, Mn2+; 10, Fe3+; 11, Li+; 12, Zr2+;
13, Sn2+; and 14, Ag+. The concentration of Hg2+ was 1 µM. The
concentrations of the other metal ions were 100 µM. Other experimental
conditions were the same as Figure 5. 35
To test the potential of the mimic sandwich ELISA method
for the analysis of Hg2+ in environmental samples, a water
sample was collected on the campus of Fudan University
(Shanghai, China). The sample was centrifuged at 12000 rpm 40
for 3 min to remove soil and other particles, and then tested
by the proposed technique. No color or CL change was
observed in this water sample, which indicates Hg2+ ions were
not detected. The water sample was then spiked with Hg2+
ions at different levels, and the recoveries of 1, 10, and 100 45
ng/mL of Hg2+ ions were 109.7±10.1 %, 102.9±2.93 % and
106.1±6.1 %, respectively. Therefore, the proposed method is
particularly attractive for monitoring very low levels of
mercury in water samples.
Conclusions 50
In summary, we have developed a mimic sandwich ELISA CL
method for the highly sensitive and selective determination of
Hg2+ ions. Compared with previous methods, this mimic
sandwich ELISA CL method has the following advantages:
(1) the mAb displays high affinity and specificity for the Hg2+ 55
ions, not the Hg-ligand complex; (2) in the mimic sandwich
immunoassay for Hg2+ ions, the CL intensity increases with
the amount of Hg2+; (3) the sensitivity is high with a detection
limit of 0.008 ppb, which is three-to-four orders of magnitude
more sensitive than many other techniques; (4) the method is 60
highly selective, which allows detection of Hg2+ ions in the
presence of an excess (100-fold) of other metal ions in
samples; (5) only a low-cost CL device is needed for detection
of Hg2+ ions, which makes the technology very attractive for
mobile and point-of-care testing; (6) the assay can be carried 65
out in 96- or 384-well plates, which are suitable for routine
high-throughput applications. This method provides a rapid,
easy, and reliable way to detect Hg2+ in environment, water,
and food samples.
70
Table 1. Comparison of sensitivity for different Hg2+ assay
methods
Analytical method Label
Detection
limit
Colorimetry Au NPs 1 nM 34
Colorimetry Terpyridine derivatives 25 µM 35
Colorimetry Hemin 4.5 nM 36
Colorimetry Hemin 50 nM 37
Fluorescence detection SYBR Green I 5 nM 38
Fluorescence detection Phthalocyanine-T conjugate 32 nM 39
Fluorescence detection Au clusters 10 nM 40
Fluorescence detection Au nanoclusters 80 nM 41
Colorimetry Boron-dipyrromethene 4.5 nM 42
UV-vis Hemin 19 nM 43
Electrochemical Glucose oxidase 100 pM44
Hyper-Rayleigh Scattering Au NPs 5 ppb
45
Colorimetry Ruthenium complexes 20 ppb46
Induced Circular Dichroism Label-free 18 nM47
Fluorescent/Colorimetric Rhodamine derivative 1 ppb48
Mimic Sandwich ELISA
(This work) Label-free
40 pM
(0.008 ppb)
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ACKNOWLEDGMENTS
Y. Z. Wang, on leave from Sichuan University, China, is
the same contributor as first author. We acknowledge
financial support from the National Drug Innovative Program 5
(2009ZX09301-011) and the National Natural Science
Foundation of China (Grant No. 21175027, 20975026,
20835003).
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Electronic Supplementary Material (ESI) for AnalystThis journal is © The Royal Society of Chemistry 2012
1
Production of Monoclonal Antibody against Mercury (II)
Ion and “Turn-on” Chemiluminescence for Mimic
Sandwich ELISA Detection of Hg2+
Ions
Sheng Caia, Yuzhen Wang
b, Deng Anping*
b, and Jianzhong Lu*
a
1School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China
2College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou,
215123, China
E-mail: [email protected]; [email protected]
Optimization of Reaction Parameters
Figure S1. CL intensity vs. the concentration of mAb. Experimental conditions: Hg2+
ion was 200
ng/mL; NH2OH, HAuCl4, luminol and AgNO3 were 5, 1, 1 and 0.2 mM, respectively. The detection
procedure was carried out as described in the Experimental section.
0
2500
5000
0 1 2 3 4 5
mAb (µg)
CL
in
ten
sity
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2
Figure S2. CL intensity vs. the concentration of the HAuCl4. Experimental conditions: mAb was 0.5 µg;
Hg2+
ion was 200 ng/mL; NH2OH, luminol and AgNO3 were 5, 1 and 0.2 mM, respectively. The
detection procedure was carried out as described in the Experimental section.
Figure S3. CL intensity vs. the concentration of NH2OH. Experimental conditions: mAb was 0.5 µg;
Hg2+
ion was 200 ng/mL; HAuCl4, luminol and AgNO3 were 0.5, 1 and 0.2 mM, respectively. The
detection procedure was carried out as described in the Experimental section.
0
3000
6000
0 0.5 1 1.5 2 2.5
HAuCl4 (mM)
CL
in
ten
sity
0
4000
8000
0 40 80 120
NH2OH (mM)
CL
in
ten
sity
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3
Figure S4. CL intensity vs. the concentration of luminol. Experimental conditions: mAb was 0.5 µg;
Hg2+
ion was 200 ng/mL; NH2OH, HAuCl4 and AgNO3 were 40, 0.5 and 0.2 mM, respectively. The
detection procedure was carried out as described in the Experimental section.
Figure S5. CL intensity vs. the concentration of AgNO3. Experimental conditions: mAb was 0.5 µg;
Hg2+
ion was 200 ng/mL; NH2OH, HAuCl4 and luminol were 40, 0.5 and 10 mM, respectively. The
detection procedure was carried out as described in the Experimental section.
0
7000
14000
0 6 12 18 24
Luminol (mM)
CL
in
ten
sity
0
10000
20000
0 0.5 1
AgNO3 (mM)
CL
in
ten
sity
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A colour graphical abstract for the contents pages:
A new monoclonal antibody against Hg2+
ions is produced and then a mimic sandwich method
is employed for the chemiluminescence detection of Hg2+
ions.
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