retraction for analystretraction for analyst: production of monoclonal antibody against mercury (ii)...

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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2 | Journal Name, [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year]

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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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

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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


0

3000

6000

0 0.5 1 1.5 2 2.5

HAuCl4 (mM)

CL

in

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0

4000

8000

0 40 80 120

NH2OH (mM)

CL

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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


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


0

7000

14000

0 6 12 18 24

Luminol (mM)

CL

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0

10000

20000

0 0.5 1

AgNO3 (mM)

CL

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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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retraction for analystretraction for analyst: production of monoclonal antibody against mercury (ii)...

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