Accepted Manuscript Title: A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe Author: Xiuxia Yu Yi He Jie Jiang Hua Cui PII: DOI: Reference:
S0003-2670(14)00067-1 http://dx.doi.org/doi:10.1016/j.aca.2014.01.021 ACA 233054
To appear in:
Analytica Chimica Acta
Received date: Revised date: Accepted date:
8-10-2013 6-1-2014 8-1-2014
Please cite this article as: X. Yu, Y. He, J. Jiang, H. Cui, A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe, Analytica Chimica Acta (2014), http://dx.doi.org/10.1016/j.aca.2014.01.021 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A competitive immunoassay for sensitive detection of small molecules
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chloramphenicol based on luminol functionalized silver nanoprobe
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Xiuxia Yu, Yi He, Jie Jiang, Hua Cui*
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CAS Key Laboratory of Soft Matter Chemistry,
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Department of Chemistry, University of Science and Technology of China,
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Hefei, Anhui 230026, P.R. China
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*Corresponding author. Prof. H. Cui,
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Tel: +86-0551-63606645
Fax: +86-0551-63600730 Email:
[email protected] 1
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Abstract
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Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad
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action spectrum against Gram-positive and Gram-negative bacteria, as
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well as anaerobes. The use of CHL is strictly restricted in poultry because
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of its toxic effect. However, CHL is still illegally used in animal farming
24
because of its accessibility and low cost. Therefore, sensitive methods are
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highly desired for the determination of CHL in foodstuffs. The
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immunoassays based on labelling as an important tool have been reported
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for the detection of CHL residues in food-producing animals. However,
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most of labelling procedures requires multi-step reactions and
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purifications and thus they are complicated and time-consuming. Recently,
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in our previous work, luminol functionalized silver nanoparticles have
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been successfully synthesized, which exhibits higher CL efficiency than luminol functionalized gold nanoparticles. In this work, the new luminol functionalized silver nanoparticles have been used for the labelling of small
molecules
CHL for
the
first
time
and
a
competitive
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chemiluminescent immunoassay has been developed for the detection of
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CHL. Owing to the amplification of silver nanoparticles, high sensitivity
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for CHL could be achieved with a low detection limit of 7.6×10-9 g mL-1
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and a wide linear dynamic range of 1.0×10-8 ~1.0×10-6 g mL-1. This 2
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method has also been successfully applied to determine CHL in milk and
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honey samples with a good recoveries (92% and 102%, 99% and 107%
41
respectively), indicating that the method is feasible for the determination
42
of CHL in real milk and honey samples. The labelling procedure is simple,
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convenient and fast, superior to previously reported labelling procedures.
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The immunoassay is also simple, fast, sensitive and selective. It is of
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application potential for the determination of CHL in foodstuffs.
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Keywords:
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Chemiluminescence; Luminol functionalized silver nanoprobe;
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Chloramphenicol;
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Immunoassay
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1. Introduction
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Chloramphenicol (CHL) as a very effective broad spectrum antibiotic
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is widely used in veterinary practice as feed additives for growth
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promotion because of its low cost. However, research has shown that it
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can lead to serious adverse reactions and side effects in humans such as
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aplastic anemia, which is often fatal [1]. These potential hazards have led
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to a prohibition of its use in the United States and European Union in
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1994 in order to protect humans from the potentially harmful antibiotic
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residues [2]. However, CHL is still illegally used in animal farming
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because of its accessibility and low cost and its residues have been found
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in various food samples, such as muscle, shrimp, milk and honey [3]. In
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order to effectively monitor the occurrence of residues of CHL, specific and sensitive analytical methods are required. In recent years, the immunoassays based on labelling as an important
tool have been reported for the detection of CHL residues in
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food-producing animals. The labelling of small molecules is very difficult
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work. A great effort has been made for labelling of CHL. Park et al. [4]
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reported a direct competitive chemiluminescent (CL) immunosensor by
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using horseradish peroxidase (HRP) as a label. The labelling procedure 4
Page 4 of 37
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was accomplished according to the activated ester method reported
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previously [5]. The CHL succinate solution first mixed with
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1-cyclohexyl-3-(2-morpholinoethyl)
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sulfonate and N-hydroxysuccinimide solution, and then HRP was added
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into the activated CHL succinate to obtaine the CHL-HRP. Zhang et al. [6]
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developed a competitive immunoassay by fluorescently labeled CHL
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hapten, using capillary electrophoresis (CE) with a laser-induced
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Fluorescence (LIF) detector. The CHL hapten was coupled to keyhole
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limpet hemocyanin (KLH) as the immunogen and was then mixed with
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fluorescein thiocarbamyl ethylenediamine (EDF) in dimethylformamide
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to form an EDF labeled CHL. Gasilova et al. [7] presented a fluorescence
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polarization immunoassay of the CHL using fluoresceinisothiocyanate
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(FITC) as a label. The labelling procedure was completed by direct
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grafting of FITC label to an aminochloramphenicol in methanol and
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carbodiimide
triethylamine blend solution. Jarujamrus et al. [8] described the feasible method for analysis of CHL using AuNPs as labels by coupling competitive immunoassay with inductively coupled plasma mass spectrometry (ICP-MS). The labelling procedure was carried out by the
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adsorption of CHL-BSA (bovine serum albumin) protein to AuNPs. Yuan
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et al. [9] reported a direct competitive gel-based visual immunoassay by
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using HRP as a label. The labelling procedure was similar with Zhang et
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al. [6] by using HRP instead of EDF to form a HRP labeled CHL. 5
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However, most of labelling procedures requires multi-step reactions and
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purifications and thus they are complicated and time-consuming.
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Therefore, it is necessary to exploit a sensitive and simple labelling
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strategy for the CHL detection. In
recent
years,
a
variety
of
CL
or
electrogenerated
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chemiluminescence (ECL) functionalized nanoparticles was synthesized
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[10-14] and has been successfully used as labels for the detection of
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macromolecules like DNA and protein [15-22]. However, the
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functionalized nanoparticles were rarely reported for labelling of small
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molecules. Recently, novel silver nanoparticles functionalized by luminol
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have been synthesized [12] by our group. In this work, they are
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successfully used for the labelling of small molecule CHL. Based on this
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luminol functionalized silver nanoprobe, a competitive immunoassay for
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sensitive detection of small molecules CHL was developed. The
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conditions for the detection of target CHL were optimized and the analytical performance of the resulting CHL immunoassay was studied. Finally, the applicability of the strategy in real milk and honey samples was explored.
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2. Experimental section
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2.1.
Chemicals and solutions 6
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AgNO3 and absolute ethanol (G, R) were purchased from Shanghai
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Reagent (Shanghai, China). Anti-chloramphenicol monoclonal antibody
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and CHL were purchased from Huaan Magnech Bio-Tech Co., Ltd. An
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AgNO3 solution of 5×10-3 mol L-1 was prepared by dissolving AgNO3 in
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purified water and stored at 4 ºC. A stock solution of luminol (1.0×10-2
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mol L-1) was prepared by dissolving luminol (Sigma) in 0.1 mol L-1
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NaOH aqueous solution. Working solutions of H2O2 were prepared fresh
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daily from 30% (v/v) H2O2 (Xin Ke Electrochemical Reagent Factory,
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Bengbu, China). Bovine serum albumin (BSA) was purchased from
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Solarbio (Beijing, China). The buffer solutions used in the experiment
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included 0.05 mol L-1 carbonate buffer (CBS, NaHCO3–Na2CO3), pH 7.4
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phosphate saline buffer (PBS). All glassware used in the following
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procedures was cleaned in a bath of freshly prepared HNO3/HCl (3:1,
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v/v), rinsed thoroughly in redistilled water, and dried prior to use.
All
other reagents were of analytical grade. Ultra-pure water was prepared by a Millipore Milli-Q system and used throughout. The food used for the analysis (milk and honey) was purchased from local supermarket.
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2.2.
Preparation of luminol-AgNPs labeled CHL
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Luminol-AgNPs were prepared by one-pot method as follows [12]: 7
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first, a 2 mL portion of AgNO3 solution with the concentration of
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5.0×10-3 mol L-1 was mixed with 5 mL ultrapure water and 9 mL absolute
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ethanol. While stirring vigorously at 25 ºC, 0.5 mL portion of luminol
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stock solution (1.0×10-2 mol L-1) were added rapidly, the solution was
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stirred for 2 h, during which time a color change from colorless to
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primrose yellow to deep yellow, indicating the formation of silver
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nanoparticles. The unreacted reagents were removed via dialysis for 2
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days with ultrapure water about six times under stirring by use of a 3500
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molecular weight cut-off dialysis membrane to obtain luminol-AgNPs.
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The morphologies of luminol-AgNPs were characterized by transmission
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electron microscope (TEM). As shown in Fig.1B, spherical nanoparticles
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with a size of approximate 25 nm were obtained.
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The luminol-AgNPs with good CL property were prepared as
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described previously and stored at 4 ºC. Luminol-AgNPs labeled CHL
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was prepared as follows: 2 mL of luminol-AgNPs colloid and 1 mL of CHL solution (1.0×10-6 g mL-1) were mixed and stirred for 40 min at room temperature for effective loading of the CHL onto the luminol functionalized silver nanoparticles. After that, a final concentration 5% of
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BSA was added into the mixed solution to block the excessive
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luminol-AgNPs, then incubated for 30 min at 37 ºC and stored at 4 ºC
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before use. A schematic for the preparation of the luminol-AgNPs labeled
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CHL is given in Fig. 1A. The morphologies of luminol-AgNPs labeled 8
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CHL were characterized by TEM as shown in Fig. 1C.
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2.3.
Immobilized antibody onto the microplate
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Coating involves the interaction between the solid phase surfaces and
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reagents in immunoassay. First, according to the introduction supplied by
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the company, each well of the microplates was washed with 100 µl of 1%
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(w/v) BSA for 5 min for three times and the plate activated with BSA
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reacted more stably and actively. Then, each well of the microplates was
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coated with 100 µl of the CHL antibody (2.0×10-6 g mL-1) in CBS buffer
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solution. The plate was allowed to stand sealed at 4 °C overnight. After
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that, the plate was washed by PBS buffer solution three times and then
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100 μl of the blocking BSA (5%) was added into each well and the plate
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was put at 37 ºC for 30 min to block the active sites on the plate.
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Subsequently, the plate was washed by PBS buffer solution in triplicate and gently tapped against tissue paper to remove all fluid and stored at 4 ºC for further use.
2.4.
Competitive immunoassay
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During the detection of target CHL, a mixture of 100 µl
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luminol-AgNPs labeled CHL solutions with 100 µl of CHL solution, after 9
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gently stirred for 10 s, was immediately added into the microplate
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modified with CHL antibody and left for incubation at 37 ºC for one hour.
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After that, the plate was washed by PBS buffer solution in triplicate and
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prepared for the further assay. CL measurements were performed with a
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microplate flow injection system. A H2O2 solution containing 0.1 mol L-1
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NaOH was used as working solution for the detection of CHL. When the
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H2O2/NaOH solution was injected into the microplate, a CL signal was
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generated and recorded.
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3. Results and discussion
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3.1. Interaction between CHL and luminol-AgNPs
It was found that CHL could conjugate with luminol-AgNPs. The
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interaction of CHL with luminol-AgNPs was studied by UV-Vis absorption spectra. Fig. 2 displays the UV-Vis absorption spectra of CHL, luminol-AgNPs and the mixture of CHL with luminol-AgNPs. A peak at about 440 nm was observed in UV-visible spectrum of luminol-AgNPs
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(Fig. 2b), which was the characteristic absorption peak of luminol-AgNPs
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due to their surface plasmon resonance [23], while only one characteristic
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absorption peak of CHL at about 270 nm in UV-visible spectrum of CHL
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(Fig. 2c) was observed. The absorption spectrum of the mixture of CHL 10
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with luminol-AgNPs (Fig. 2e) was not equal to the sum of the absorbance
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of CHL and luminol-AgNPs with same concentrations, respectively (Fig.
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2d), which indicated that there was some interaction between CHL and
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luminol-AgNPs. After reaction between CHL and luminol-AgNPs, the
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solution was centrifuged and the precipitates were redispersed in PBS
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buffer to remove the unreacted CHL. UV-Vis absorption spectrum of
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redispersed solution is shown in Fig. 2f. Obviously, the characteristic
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absorption peaks of luminol-AgNPs and CHL still could be observed so
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that CHL was immobilized on the surface of luminol-AgNPs.
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CHL is an organic molecule having amide and carbonyl groups. Patil
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et al. [24] reported that CHL with an amide functional group has great
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potential to form hydrogen bond with a carbonyl functional group of
217
poly(vinylpyrrolidone)
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nanoparticles, giving rise to CHL-loaded sliver nanoparticles against
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Salmonella typhi. Luminol molecules on the surface of silver nanoparticles contain carbonyl group. Accordingly, we deduced that amide group −NH in CHL molecules might interact with carbonyl group −C=O in luminol molecules to form hydrogen bond, resulting in formation of CHL-conjugated luminol-AgNPs.
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3.2. CL behavior of the immunosensor
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A schematic diagram of competitive immunoassay for CHL is 11
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illustrated in Fig. 3. The CL behavior of the CHL immunosensor was
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studied by injecting 1.0×10-4 mol L-1 H2O2 solution containing 0.1 mol L-1
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NaOH into the microplate containing immunoconjugates. Under the
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different stages, the CL signal was recorded as shown in Fig. 4. Curves
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a-b show the CL responses on a bare microplate, on an antibody modified
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microplate. As we can see, a weak CL signal was observed on these
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microplates, which might be caused by background. In contrast, a strong
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CL signal was observed as shown in curve c of Fig. 4 on the microplate
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containing immunoconjugates. These results indicated that only the
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antibody modified microplate reacted with the luminol-AgNPs labeled
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CHL could produce CL signals and the CL signal was from
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luminol-AgNPs. The CL signal was ascribed to that AgNPs could
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catalyze the decomposition of H2O2 to produce some reactive
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intermediates such as hydroxyl radical, superoxide anion. Hydroxyl
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radical reacted with luminol to form luminol radical and diazaquinone, followed by the reaction with superoxide anion or monodissociated hydrogen peroxide, giving rising to light emission [25].
3.3. Optimization of experimental conditions
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To obtain an optimal CL signal, the effects of H2O2 concentration and
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NaOH concentration on the CL intensity were investigated. Meanwhile, 12
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incubation time for the CHL and luminol-AgNPs interaction greatly
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influenced the sensitivity of the developed immunoassay so the
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incubation time for the CHL and luminol-AgNPs was also examined.
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3.3.1. Effect of H2O2 concentration
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The change of CL intensity with the concentration of H2O2 is shown
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in Fig. 5. It can be seen that the CL intensity markedly enhanced with
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increasing the concentration of H2O2 at the beginning. When the H2O2
258
concentration reached 1.0×10-4 mol L-1, the CL intensity reached a
259
maximum. When the H2O2 concentration was higher than 1.0×10-4 mol L-1,
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the CL intensity did not change obviously. In the CL reaction, H2O2 first
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reacted with OH- to produce hydroxyl radical OH.-, which followed by
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the reaction with luminol anion via multi-steps to yield CL emission
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[26-28]. Thus the CL intensity increased with increasing the H2O2
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concentration at the beginning. When the H2O2 concentration was over certain value, luminol may be exhausted, leading to almost constant CL intensity. Therefore, 1.0×10-4 mol L-1 H2O2 was adopted in the following experiments.
3.3.2. Effect of NaOH concentration
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The CL intensity of luminol depends on pH of the solution [29]. Thus
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the effect of NaOH concentration in the range of 0.0 mol L-1 – 1.0 mol L-1
271
was examined. As shown in Fig. 6, with increasing concentration of 13
Page 13 of 37
NaOH solution, the CL intensity increased greatly in the range of 0.0 -
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0.1 mol L-1. When the NaOH concentration was over 0.1 mol L-1, the CL
274
intensity increased slighly. When the NaOH concentration was over 0.5
275
mol L-1, the CL intensity decreased. It may be due to that higher NaOH
276
concentration promoted the dissociation of H2O2 to form more hydroxyl
277
radical, leading to an increase in CL intensity at the beginning. When the
278
NaOH concentration was over 0.1 mol L-1, other reactant in the CL
279
reaction may be exhausted and thus the CL intensity did not change
280
obviously. When the NaOH concentration was over 0.5 mol L-1, the color
281
of the luminol-AgNPs changed immediately from deep yellow to grey
282
after adding NaOH and obvious precipitates were observed, resulting in a
283
decrease in CL intensity. Thus 0.1 mol L-1 NaOH was employed for the
284
following experiments.
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3.3.3. Effect of reaction time of CHL and luminol-AgNPs
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The effect of reaction time of CHL and luminol-AgNPs on the CL
intensity was studied in the range of 0 - 60 min as shown in Fig. 7. The CL intensity showed a positive correlation with the reaction time in 40 min, which should be attributed to more effective binding between CHL
290
and luminol-AgNPs in the initial period. However, when the reaction time
291
was longer than 40 min, the CL intensity went down oppositely, which
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probably caused by the coagulation of luminol-AgNPs, which was
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confirmed in our experiment that the luminol-AgNPs trended to be 14
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coagulated on the condition of a long reaction time with CHL or a high
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concentration of CHL. Thus, 40 min was chosen as the best reaction
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time.
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3.4 Analytical performance
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Under the optimized conditions, it was found that the ratio between
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the CL intensity of CHL and the CL intensity of the blank solution
302
decreased linearly with the increment of the concentration of CHL
303
(shown in Fig. 8) in the range of 1.0×10-8 -1.0×10-6 g mL-1. The
304
regression equation was I/I0 = -0.030 – 0.118×log C and the correlation
305
coefficient was 0.994, where I/I0 was the ratio between the CL intensity
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of CHL and the CL intensity of the blank solution and C was the
307
concentration of target CHL in g mL-1. The detection limit for target CHL
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at a signal-to-noise ratio of 3 (S/N = 3) was 7.6×10-9 g mL-1. In addition, the detection limit (3σ/k) according to international union of pure and applied chemistry of the immunoassay for CHL was determined to be 2.0 ×10-8 g mL-1. Additionally, 1.0×10−6 g mL-1 CHL solution was determined
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with five replicate determinations under the optimized conditions, and the
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relative standard deviations for the CL intensity are 3.26%. A comparison
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between the proposed CL immunoassay and other previously reported
315
immunoassays for the assay of CHL is listed in Table 1. The results 15
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showed that the limit of detection of the presented CL immunoassay is
317
comparable with some previously reported methods for the determination
318
of CHL [4, 6-9, 30-32] and is higher than that of the immunoassays based
319
on CE-LIF [6], ICP-MS [8], Gel-based visual immunoassay [9] and
320
lateral flow immunoassay [32]. However, as you can also see in Table 1,
321
most of labelling procedures requires multi-step reactions and
322
purifications and thus they are complicated and time-consuming.
323
Moreover, the measurement also involved complex and expensive
324
instrumentation. In contrast, the labelling procedure of the proposed
325
method is simple and fast and the detection does not need complex and
326
expensive instrumentation, making the method low-cost, easy to operate
327
and time-saving. The results demonstrated that this method could be used
328
for the sensitive determination of the target CHL. The result also revealed
329
that AgNPs bound with luminol were of excellent labelling property for
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CHL.
3.5 Specificity of the CHL immunoassay
334
To affirm the specificity of the CHL immunosensor, we chose
335
tetracycline, streptomycin, neomycin, ciprofloxacin and ofloxacine as
336
interferents to evaluate the specificity of the immunosensor. As can be
337
seen in Fig. 9, tetracycline, streptomycin, neomycin, ciprofloxacin and 16
Page 16 of 37
ofloxacine with same concentration (1.0×10-6 g mL-1) were used for
339
immunoassays instead of CHL, the CL responses were comparable with
340
the blank, and almost negligible compared with the CHL. Moreover, the
341
CL response of CHL was not significantly affected in the case of
342
interferences exist, compared with CHL in the system only.
cr 3.6 Detection of CHL spiked in real samples
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The proposed competitive immunoassay for sensitive detection of
347
small molecules CHL was used to analyze CHL in the milk and honey
348
samples obtained from a market in China. A potion of 1.0 mL milk and
349
1.0 mL honey were accurately measured and diluted 10 times with PBS
350
buffer. The mixtures were placed in a water bath at 60 °C for 5 min and
351
then be vortexed until a homogeneous sample was obtained, and then
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each sample was filtered by ultrafiltration tubes to obtain the transparent solution in the tube. Known concentrations of standard CHL (5.0×10−8, 1.0×10−7 and 5.0×10−7 g mL-1) were added to the samples and determined by the immunosensor. The results are summarized in Table 2. The
356
recoveries of the CHL standards in milk sample are 92 % and 102 %,
357
respectively, while 99 % and 107 % in honey sample, respectively. These
358
results indicated that the proposed immunosensor can be used to
359
determine CHL in the milk and honey samples. 17
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4. Conclusion We have developed a competitive immunoassay for sensitive
362
detection of small molecules CHL using the luminol functionalized silver
363
nanoparticles as nanoprobe for the first time. CHL in a range of 1.0×10-8
364
-1.0×10-6 g mL-1 could be detected by use of the CL intensity with a low
365
detection limit of 7.6×10-9 g mL-1. The labelling procedure is simple and
366
fast, superior to all the previously reported labelling procedures [4, 6-8].
367
Since the labelling of small molecules is very difficult work, the present
368
work provides a novel and effective labelling strategy for CHL. The
369
immunoassay is simple, fast, cost-effective, sensitive and selective. The
370
simplicity of the assay and the lack of requirement for sophisticated
371
equipment render the CHL immunoassay a promising candidate as a rapid
372
test for the detection of CHL in practical applications.
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the Fundamental Research Funds for the Central Universities (Grant
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Nos.WK2060190007) and the Overseas Outstanding Young Scientist
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Program of the Chinese Academy of Sciences.
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Acknowledgements
This work was supported by the National Natural Science Foundation of P. R. China (Grant Nos. 21173201, 21075115, 20625517 and 20573101),
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Page 21 of 37
438 439
Table 1. A comparison of the proposed CL immunoassay with other previously reported immunoassays for the assay of CHL Protocol
Labelling procedure
Detection
Linear
method
range
Detection limit -1
CL immunosensor
(ng mL )
(ng mL-1)
CL
-
3.23
CE-LIF
0.008–5
0.0016
CHL+EDF
Overnight in the dark
2h Purify Thin-layer chromatography
linked
immunoassay
CHL-BSA+AuNPs Centrifuge
Stir; room temperature
d
CHL+protein Purify
Assay
te
Enzyme-Linked
10–3000
4.52
8
Visual detection
-
1
9
enzyme-linked
0.068–3.92
0.042
30
electrochemical
0.05–0.95
0.045
31
lateral-flow
-
0.2
32
7.6
This work
Overnight in the dark
Filter
Immunosorbent
0–20
Redisperse
Gel-based visual CHL+HRP
7
ICP-MS
Mix 30 min
15 min
immunoassay
10
polarization
an
Immunoassay
ICP-MS
fluorescence Stir; room temperature
CHL+FITC
M
Polarization
us
Filter 5 days Fluorescence
6
cr
immunoassay
Stir; room temperature
4
ip t
Stir; room temperature CHL+HRP 2h Incubate overnight Dialyze 5 days 4 ºC CE-LIF
Reference
NHS a and DCC b Overnight
immunosorbent assay
Ac ce p
Silica gel column
Amperometric
immunosensor
CHL+hydrazine Dialyze 24 hours
Amide reaction Overnight
Filter
Quantum
dot-based
lateral
CHL antibody+quantum dots
flow immunoassay
Stir; room temperature
assay
90 min in the dark
Five 15-min centrifugations
CL immunoassay
CL
10–1000
CHL+luminol-AgNPs Mix for 40 min Room temperature
440 441
a
NHS: N-hydroxysuccinimide b DCC: N,N-dicyclohexyl carbodiimide
442 443 22
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443 444
Table 2. Analytical performance of competitive immunoassay for CHL
446
spiked in milk and honey samples
ip t
445
cr
447
451 452 453
us
94.4
1.8
52
104
1.3
107
2.6
485
99
1.6
46
92
2.1
100
102
500
472
50 100
M
an
50
107
te
milk
(ng mL-1)
Ac ce p
450
1.3
Recovery (%)
Spiked
500 449
102
Found (ng mL-1)
Sample
honey
CV (%)
d
448
454 455
23
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455 456
Figures
457
Fig. 1 A schematic (A) for the preparation of the CHL-Luminol-AgNPs.
459
TEM images of luminol-AgNPs before (B) and after (C) the conjugation
460
with CHL.
us
cr
ip t
458
461
Fig. 2 UV-Vis absorption spectra. (a) Blank; (b) luminol-AgNPs; (c)
463
2.0×10-5 g mL-1 CHL; (d) b+c; (e) luminol-AgNPs reacted with CHL with
464
the same concentrations as b and c; (f) mixture of CHL with
465
luminol-AgNPs after centrifugation.
d
M
an
462
te
466
468 469 470 471
Ac ce p
467
Fig. 3 Schematic diagram of the proposed CHL detection strategy.
Fig.4 CL signals were obtained (a) on a bare microplate, (b) on a antibody
modified
microplate, (c)
on
a
microplate
containing
472
immunoconjugates fromed between CHL-luminol-AgNPs and antibody.
473
All CL signals were measured in 1.0×10-4 mol L-1 H2O2 solution
474
containing 0.1 mol L-1 NaOH.
475
24
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476
Fig. 5 Effect of H2O2 concentration on the CL intensity. NaOH, 0.1 mol
478
L-1.
ip t
477
479
cr
480
Fig. 6 Effect of NaOH concentration on the CL intensity. H2O2, 1.0×10-4
482
mol L-1.
us
481
an
483 484
Fig. 7 Effect of reaction time of CHL and luminol-AgNPs on the CL
486
intensity. CL signals were measured in 1.0×10-4 mol L-1 H2O2 containing
487
0.1 mol L-1 NaOH solution.
490 491 492 493
d
te
489
Ac ce p
488
M
485
Fig. 8 Calibration curve of CL intensity of target CHL. H2O2, 1.0×10-4 mol L-1; NaOH, 0.1 mol L-1.
Fig. 9 Specificity of the CHL immunoassay. a: blank;b: 1.0×10-6 g mL-1
494
tetracycline ; c: 1.0×10-6 g mL-1 streptomycin ; d: 1.0×10-6 g mL-1
495
neomycin ; e: 1.0×10-6 g mL-1 ciprofloxacin ; f: 1.0×10-6 g mL-1
496
ofloxacine;g: 1.0×10-6 g mL-1 CHL;h: 1.0×10-6 g mL-1 CHL with
497
1.0×10-6 g mL-1 tetracycline, streptomycin , neomycin, ciprofloxacin and 25
Page 25 of 37
498
ofloxacine.
499
ip t
500 501
Ac ce p
te
d
M
an
us
cr
502
26
Page 26 of 37
502 503
ip t
504
506 507 508 509 510
Ac ce p
te
d
M
an
us
cr
505
Fig. 1
511 512 513 514 27
Page 27 of 37
515 516
ip t
517 518
cr
519
521 522 523 524
Ac ce p
te
d
M
an
us
520
525 526 527
Fig. 2
528 28
Page 28 of 37
528 529
an
us
cr
ip t
530
M
531
535
te
534
Fig. 3
Ac ce p
533
d
532
29
Page 29 of 37
535 536
540 541 542 543 544
te
539
Ac ce p
538
d
M
an
us
cr
ip t
537
Fig. 4
545
30
Page 30 of 37
545
549 550 551 552
te
548
Fig. 5
Ac ce p
547
d
M
an
us
cr
ip t
546
31
Page 31 of 37
552 553
ip t
554 555
557 558 559 560 561
Ac ce p
te
d
M
an
us
cr
556
Fig. 6
562
32
Page 32 of 37
562
566 567 568 569 570 571
te
565
Ac ce p
564
d
M
an
us
cr
ip t
563
Fig. 7
572
33
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572 573
ip t
574 575
577 578 579 580 581
Ac ce p
te
d
M
an
us
cr
576
Fig. 8
582 583 584 585 34
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585
589 590 591 592 593
te
588
Ac ce p
587
d
M
an
us
cr
ip t
586
Fig. 9
35
Page 35 of 37
ip t cr Ac ce p
te
d
M
an
us
594 595
36
Page 36 of 37
te
d
M
an
us
cr
ip t
Highlights Novel silver nanoparticles functionalized by luminol are used for the labeling. The labelling procedure is simple, convenient and fast. A competitive CL immunoassay has been developed for the detection of CHL. The immunoassay is simple, fast, sensitive and selective. It is of application potential for the determination of CHL in foodstuffs.
Ac ce p
595 596 597 598 599 600 601 602
37
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