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]

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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

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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%

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respectively), indicating that the method is feasible for the determination

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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

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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

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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.

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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

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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

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concentration reached 1.0×10-4 mol L-1, the CL intensity reached a

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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

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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

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intensity increased slighly. When the NaOH concentration was over 0.5

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mol L-1, the CL intensity decreased. It may be due to that higher NaOH

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concentration promoted the dissociation of H2O2 to form more hydroxyl

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radical, leading to an increase in CL intensity at the beginning. When the

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NaOH concentration was over 0.1 mol L-1, other reactant in the CL

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reaction may be exhausted and thus the CL intensity did not change

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obviously. When the NaOH concentration was over 0.5 mol L-1, the color

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of the luminol-AgNPs changed immediately from deep yellow to grey

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after adding NaOH and obvious precipitates were observed, resulting in a

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decrease in CL intensity. Thus 0.1 mol L-1 NaOH was employed for the

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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

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and luminol-AgNPs in the initial period. However, when the reaction time

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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

Page 14 of 37

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

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decreased linearly with the increment of the concentration of CHL

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(shown in Fig. 8) in the range of 1.0×10-8 -1.0×10-6 g mL-1. The

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regression equation was I/I0 = -0.030 – 0.118×log C and the correlation

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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

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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

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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

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comparable with some previously reported methods for the determination

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of CHL [4, 6-9, 30-32] and is higher than that of the immunoassays based

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on CE-LIF [6], ICP-MS [8], Gel-based visual immunoassay [9] and

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lateral flow immunoassay [32]. However, as you can also see in Table 1,

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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.

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Moreover, the measurement also involved complex and expensive

324

instrumentation. In contrast, the labelling procedure of the proposed

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method is simple and fast and the detection does not need complex and

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expensive instrumentation, making the method low-cost, easy to operate

327

and time-saving. The results demonstrated that this method could be used

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for the sensitive determination of the target CHL. The result also revealed

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that AgNPs bound with luminol were of excellent labelling property for

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CHL.

3.5 Specificity of the CHL immunoassay

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To affirm the specificity of the CHL immunosensor, we chose

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tetracycline, streptomycin, neomycin, ciprofloxacin and ofloxacine as

336

interferents to evaluate the specificity of the immunosensor. As can be

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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

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immunoassays instead of CHL, the CL responses were comparable with

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the blank, and almost negligible compared with the CHL. Moreover, the

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CL response of CHL was not significantly affected in the case of

342

interferences exist, compared with CHL in the system only.

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The proposed competitive immunoassay for sensitive detection of

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small molecules CHL was used to analyze CHL in the milk and honey

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samples obtained from a market in China. A potion of 1.0 mL milk and

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1.0 mL honey were accurately measured and diluted 10 times with PBS

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buffer. The mixtures were placed in a water bath at 60 °C for 5 min and

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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

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recoveries of the CHL standards in milk sample are 92 % and 102 %,

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respectively, while 99 % and 107 % in honey sample, respectively. These

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results indicated that the proposed immunosensor can be used to

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determine CHL in the milk and honey samples. 17

Page 17 of 37

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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

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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

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fast, superior to all the previously reported labelling procedures [4, 6-8].

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Since the labelling of small molecules is very difficult work, the present

368

work provides a novel and effective labelling strategy for CHL. The

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immunoassay is simple, fast, cost-effective, sensitive and selective. The

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simplicity of the assay and the lack of requirement for sophisticated

371

equipment render the CHL immunoassay a promising candidate as a rapid

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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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References

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[1]

E.H. Allen, J. Assoc. Off. Anal. Chem. 68 (1985) 990.

383

[2]

A.A.M. Stolker, U. A. T. Brinkman, J. Chromatogr. 1067 (2005) 15.

384

[3]

J. Xu, W. Yin, Y. Zhang, J. Yi, M. Meng, Y. Wang, H. Xue, T.

ip t

381

cr

Zhang, R. Xi, Food Chem. 134 (2012) 2526.

385

[4]

I.S. Park, N. Kim, Anal. Chim. Acta 578 (2006) 19.

387

[5]

A.Y. Kolosova, J.V. Samsonova, A.M. Egorov, Food Agric.

[6]

C. Zhang, S. Wang, G.Z. Fang, Y. Zhang, L.Q. Jiang,

M

Electrophoresis 29 (2008) 3422.

390

an

Immunol. 12 (2000) 115.

388 389

us

386

391

[7]

N.V. Gasilova, S.A. Eremin, J. Anal. Chem. 65 (2010) 255.

392

[8]

P.

396 397 398 399 400

d

Chawengkirttikul,

J.

Shiowatana,

A.

te

[9]

M. Yuan, W. Sheng, Y. Zhang, J.P. Wang, Y.J. Yang, S.G. Zhang,

Ac ce p

395

R.

Siripinyanond, J. Anal. At. Spectrom. 27 (2007) 884.

393 394

Jarujamrus,

I.Y. Goryacheva, S. Wang, Anal. Chim. Acta 751 (2012) 128.

[10] H. Cui, W. Wang, C.F. Duan, Y.P. Dong, J.Z. Guo, Chem. Eur. J. 13 (2007) 6975.

[11] W. Wang, H. Cui, J. Phys. Chem. C 112 (2008) 10759. [12] Y. He, D.Q. Liu, X.Y. He, H. Cui, Chem. Commun. 47 (2011) 10692.

401

[13] Y.Q. Yu, M. Zhou, H. Cui, J. Mater. Chem. 21 (2011) 12622.

402

[14] D.Y. Tian, Y. Chai, H.L. Zhang, H. Cui, Chem. Commun. 47 (2011) 19

Page 19 of 37

407 408 409 410 411

[16] N. Li, J.Z. Guo, B. Liu, Y.Q. Yu, H. Cui, L.Q. Mao, Y.Q. Lin, Anal.

ip t

406

Talanta 78 (2009) 399.

Chim. Acta 645 (2009) 48.

cr

405

[15] D.Y. Tian, C.F. Duan, W. Wang, N. Li, H. Zhang, H. Cui, Y.Y. Lu,

[17] Y. Chai, D.Y. Tian, W. Wang, H. Cui, Chem. Commun. 46 (2010) 7560.

us

404

4959.

[18] D.Y. Tian, C.F. Duan, W. Wang, H. Cui, Biosens. Bioelectron. 25

an

403

(2010) 2290.

[19] J. Jiang, Y. Chai, H. Cui, RSC Adv. 1 (2011) 247.

413

[20] W. Shen, D.Y. Tian, H. Cui, D. Yang, Z.P. Bian, Biosens. Bioelectron. 27 (2011) 18.

d

414

M

412

[21] Y. Chai, D.Y. Tian, J. Gu, H. Cui, Analyst 136 (2011) 3244.

416

[22] Y. Chai, D.Y. Tian, H. Cui, Anal. Chim. Acta 715 (2012) 86.

418 419 420 421 422

Ac ce p

417

te

415

[23] S. Li, Y. Shen, A. Xie, X. Yu, L. Qiu, L. Zhang, Q. Zhang, Green Chem. 9 (2007) 852.

[24] S. S. Patil, R. S. Dhumal, M. V. Varghese, A. R. Paradkar, P. K. Khannal, Synth. React. Inorg. M 39 (2009) 65.

[25] J.Z. Guo, H. Cui, W. Zhou, W. Wang, J. Photochem. Photobiol. A 193 (2008) 89.

423

[26] A. Mer´enyi, J.S. Lind, J. Am. Chem. Soc. 102 (1980) 5830.

424

[27] A.L. Rose, T.D. Waite, Anal. Chem. 73 (2001) 5909. 20

Page 20 of 37

430 431 432 433 434

[30] L. Wang, Y. Zhang, X. Gao, Z.J. Duan, S. Wang, J. Agric. Food Chem. 58 (2010) 3265.

[31] D.M. Kim, M.A. Rahman, M. H. Do, C. Ban, Y.B. Shim, Biosens. Bioelectron. 25 (2010) 1781.

[32] A.R. Berlina, N.A. Taranova, A.V. Zherdev, Y.Y. Vengerov, B.B. Dzantiev, Anal. Bioanal. Chem 405 (2013) 4997.

te

438

Ac ce p

437

d

435 436

ip t

429

Chem. C 111 (2007) 4561.

cr

428

[29] C.F. Duan, H. Cui, Z.F. Zhang, B. Liu, J.Z. Guo, W. Wang, J. Phys.

us

427

(1990) 53.

an

426

[28] G. Mer´enyi, J. Lind, T.E. Eriksen, J. Biolumin. Chemilumin. 5

M

425

21

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

Page 22 of 37

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

Page 23 of 37

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

Page 24 of 37

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

Page 33 of 37

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

Page 34 of 37

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

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594 595

36

Page 36 of 37

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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

Page 37 of 37

A competitive immunoassay for sensitive detection of small molecules chloramphenicol based on luminol functionalized silver nanoprobe.

Chloramphenicol (CHL) as a broad-spectrum antibiotic has a broad action spectrum against Gram-positive and Gram-negative bacteria, as well as anaerobe...
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