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Food Chemistry 131 (2012) 1021–1025
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier .com/locate / foodchem
Analytical Methods
Immunomagnetic reduction assay on chloramphenicol extracted from shrimp
S.Y. Yang a,b, C.S. Ho b, C.L. Lee a, B.Y. Shih a, H.E. Horng a,⇑, Chin-Yih Hong c,⇑, H.C. Yang d,⇑,Y.H. Chung e, J.C. Chen e, T.C. Lin f
a Institute of Electro-optical Science and Technology, National Taiwan University, Taipei 116, Taiwanb MagQu Co., Ltd., Sindian City, Taipei County 231, Taiwanc Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwand Department of Physics, National Taiwan University, Taipei 106, Taiwane Department of Food and Nutrition, Meiho University of Technology, Neipu, Pingtung County, Taiwanf Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung County, Taiwan
a r t i c l e i n f o
Article history:Received 12 February 2010Accepted 19 September 2011Available online 29 September 2011
Keywords:Immunomagnetic reductionMagnetic nanoparticlesChloramphenicol
0308-8146/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.foodchem.2011.09.064
⇑ Corresponding authors. Addresses: Institute ofTechnology, National Taiwan University, Taipei 116,Co., Ltd., Sindian City, Taipei County 231, TaiwanPhysics, National Taiwan University, Taipei 106, Taiw
E-mail addresses: [email protected] (H.E.u.edu.tw (C.-Y. Hong), [email protected] (H.C.
a b s t r a c t
The application of the assay methodology, called immunomagnetic reduction, using bio-functionalizedmagnetic nanoparticles as labeling markers for chloramphenicol was investigated. The reduction in thealternative-current (ac) magnetic susceptibility vac of magnetic nanoparticles caused by the associationbetween magnetic nanoparticles and chloramphenicol was detected as a function of the concentration ofchloramphenicol. In this study, the characterizations used to detect chloramphenicol, such as low-detection limit and interference, were also conducted. Furthermore, the extracting processes forchloramphenicol from shrimp were explored. Thus, the platform for detecting chloramphenicol residuein shrimp via immunomagnetic reduction was demonstrated. Such platform showed features of a0.1-ppb low-detection limit, low interference from other kinds of antibiotics, and easy operation.
� 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Chloramphenicol (CAP), a bacteriostatic antimicrobial that orig-inally found as a product of the metabolism of the soil bacteriumStreptomyces venezuelae (order Actinomycetales), is now synthe-sized chemically. It achieves its antibacterial effect by interferingwith protein synthesis in micro-organisms. Thus, CAP seems goodfor shrimp or fish feed. However, CAP may lead to bone marrowsuppression, leukemia, and aplastic anemia in human beings.Hence, the use of CAP is strictly controlled. The permitted concen-tration of CAP is 0.3 ng in 1-g of fish or shrimp, i.e. 0.3 ppb.
At present, several methods are popularly applied to determinethe concentration of CAP in aquatic food. Liquid chromatography/tandem mass spectrometry (LC/MS/MS) is the most popular plat-form for detecting CAP because of its high sensitivity and highspecificity (Ramos et al., 2003). However, any sample detectedwith LC/MS/MS must be very pure. In such cases, the extractionprocesses for CAP from aquatic food are complicated, time con-suming, and expensive. This results in the difficulties for widely
ll rights reserved.
Electro-optical Science andTaiwan (H.E. Horng), MagQu(C.-Y. Hong), Department ofan (H.C. Yang).Horng), [email protected]).
spreading LC/MS/MS to determine CAP on fields. Consequently,LC/MS/MS is currently only used at central laboratories.
Instead of using LC/MS/MS, people may use high performanceliquid chromatography (HPLC) or competitive enzyme-linkedimmunosorbent assay (ELISA) because of its inexpensive analyzersand easy operation for analyzing CAP residue in aquatic food.Hence, HPLC or competitive ELISA is much more popular comparedto LC/MS/MS in rapid assay. However, both of HPLC and competi-tive ELISA show disadvantages, such as high interference origi-nated from sample colour, variations in ion concentrations ofsamples. Therefore, researchers have kept exploring alternativetechnologies, e.g. electrophoresis (Jin, Ye, Yu, & Dong, 2000), sur-face plasma resonance (Ferguson et al., 2005), chemiluminescence(Icardo, Misiewicz, Ciucu, Mateo, & Calatayud, 2003; Park & Kim,2006), for analyzing CAP in aquatic food easily and accurately.These methodologies involve measurements of optical intensity,related to the concentrations of CAP. In this work, a technologyinvolving magnetic measurement, referred to as immunomagneticreduction (IMR) (Hong et al., 2006), was developed to determinethe concentration of CAP.
IMR is a method of assaying target molecules via measuring thereduction in the mixed-frequency magnetic susceptibility of mag-netic reagent owing to the association between bio-functionalizedmagnetic nanoparticles and target molecules (e.g. CAP in case), asillustrated in Fig. 1a and b. Under external multiple ac magneticfields, magnetic nanoparticles oscillate with the multiple ac
(a)
(b)
Fig. 1. Illustration of mechanism of immunomagnetic reduction to detect bio-targets. (a) Each magnetic nanoparticle oscillates individually with the appliedalternative-current magnetic field before binding with bio-targets. (b) Portion ofmagnetic nanoparticles become larger due to the association with bio-targets.These associated magnetic nanoparticles in (b) contribute to the reduction in thealternative-current magnetic susceptibility of reagent.
1022 S.Y. Yang et al. / Food Chemistry 131 (2012) 1021–1025
magnetic fields via magnetic interaction. Thus, the reagent underexternal multiple ac magnetic fields shows a magnetic property,called mixed-frequency ac magnetic susceptibility vac, as illus-trated in Fig. 1a. Through the anti-bodies on the outmost shell,magnetic nanoparticles associate with bio-targets. With the associ-ation, magnetic nanoparticles become larger, as schematicallyshown in Fig. 1b. The response of these larger magnetic nanoparti-cles to external multiple ac magnetic fields becomes weaker thanthat of originally individual magnetic nanoparticles. Therefore,the vac of the magnetic reagent is reduced due to the associationbetween magnetic nanoparticles and bio-targets. In principle, themore amounts of bio-targets mix with a reagent, the more mag-netic nanoparticles become clustered, thus, larger reduction invac of reagents can be expected.
According to the description given above, IMR exhibits severalunique advantages. Firstly, the unbound bio-targets and magneticnanoparticles are not necessary to be removed, therefore, the assayprocesses of IMR become simple. Secondly, only one kind of anti-body is used, thus, reduces the cost of reagent. Thirdly, IMR is a di-rect and homogeneous assay, which usually shows high reliabilityand sensitivity. Fourthly, because the amount of reduction in vac
can be accurately measured corresponding to the concentrationof bio-targets, the concentration of bio-targets can thus be mea-sured quantitatively.
The detections of proteins and viruses using IMR have beendemonstrated (Hong et al., 2007; Yang, Chieh, et al., 2008; Yang,Jian, et al., 2008). It was pointed out that there exists a very lowinterference from sample colour or other material in a matrix forIMR (Hong et al., 2007; Yang, Chieh, et al., 2008; Yang, Jian, et al.,2008; Yang et al., 2010). However, the feasibility of analyzingchemicals such as CAP using IMR is not clear. In this work, themethod for analyzing CAP using IMR is explored. Meanwhile, theapplication of IMR on CAP residual in shrimp is investigated. Todo this, it is necessary to develop the extraction processes forCAP residue from shrimp for IMR. The details of reagent, extractionprocesses, and assay characteristics and validation are described.
2. Materials and methods
2.1. Synthesis of reagent
The protocol of synthesizing magnetic Fe3O4 nanoparticles wasproposed by MagQu Co., Ltd. A ferrite solution containing a stoichi-ometric ratio of 1:2 ferrous sulphate hepta-hydrate (FeSO4�7H2O)and ferric chloride hexa-hydrate (FeCl3�6H2O) was mixed with anequal volume of aqueous dextran, which acted as a surfactant forFe3O4 particles dispersed in water. The mixture was heated to70–90 �C and titrated with strong base solution to form blackFe3O4 particles. Aggregates and excess unbound dextran were re-moved by centrifugation and gel filtration chromatography to ob-tain highly concentrated homogeneous magnetic fluid. Thereagent with desired magnetic concentration was obtained bydiluting the highly concentrated magnetic fluid with pH-7.4 phos-phate buffered saline (PBS) solution.
To make anti-bodies, i.e. anti-CAP (Ab35658-500, Abcam),bound to the dextran on the outmost shell of magnetic nanoparti-cles, NaIO4 solution was added into the magnetic solution to oxidedextran, which was then used to create aldehyde groups (ACHO).Then, dextran can react with anti-CAP via the linking of [email protected], anti-CAP is bound covalently to dextran. Through magneticseparation, unbound anti-CAP was separated from the solution.By using dynamic laser scattering, the size distribution of magneticnanoparticles bio-functionalized with anti-CAP was analyzed.
2.2. IMR detection
Magnetic reagent (40 ll) is thoroughly mixed with 60 ll samplesolution in a glass tube. The concentration of magnetism in the re-agent used for IMR detection is 0.1 emu/g (=5 mg Fe/ml). The vac
signal, vac,o, of the mixture before the formation of immuno com-plex of CAP-anti-CAP-dextran-magnetic-nanoparticle was re-corded by using a magnetic immunoassay analyzer (XacPro-E101,MagQu). Then, the mixture was kept at room temperature for theformation of CAP-anti-CAP-dextran-magnetic-nanoparticle, fol-lowed by recording the vac signal, vac,/, of the mixture. With themeasured vac,o and vac,/, the IMR signal can be obtained via thebelow:
IMR ð%Þ ¼ ðvac;o � vac;/Þ=vac;o � 100% ð1Þ
For a sample solution of given CAP concentration, the time-dependence vac signal is detected triply.
2.3. Extraction processes for CAP from shrimp
A convenient extraction process for CAP in shrimp was devel-oped in this work. After removing shells, several shrimps were pul-ped for 10 min. To extract CAP in pulped shrimp, 6 ml extractionsolution which is mainly consisted of ethyl acetate was mixed with10 g pulped shrimp. Then, the liquid was separated from the pul-ped shrimp by using a centrifuge at 5000 rpm for 5 min. A portionof CAP, originally residual in the pulped shrimp, was extracted intothe separated liquid. To enhance the extract efficiency, the extract-ing step for CAP from pulped shrimp is repeated two more times.The collected liquid containing extracted CAP was then dried byevaporating the liquid with the aid of pumping accessories. Thedried CAP was dissolved into 1-g diluent, which consists of di-methyl sulfoxide (DMSO) and PBS, to obtain a sample solution. Itshould be noted that the time spent for the extraction processesdeveloped in this work is just one third of the conventional extrac-tion processes for CAP analysis using LC/MS/MS. Furthermore, theinstruments used for processes developed in this work are simplercompared to those for extraction processes using LC/MS/MS.
S.Y. Yang et al. / Food Chemistry 131 (2012) 1021–1025 1023
Conventionally, extraction processes usually use a vacuum evapo-rator at the step of drying extracted CAP solution for LC/MS/MS. In-stead, an inexpensive and easy acquirable pump is used at the stepof drying extracted CAP solution. Therefore, the extraction pro-cesses used here are user-friendly and can easily be promoted forindustrial applications.
3. Results and discussion
3.1. Characterizations of reagent
The size distribution of the magnetic nanoparticles bio-func-tionalized with anti-CAP was analyzed with dynamic laser scatter-
(a)
Anti-CAP
40 80 120 160Diameter (nm)
0
10
20
Frac
tion
(%)
(b)
FITC
Anti-CAP
(c) (d)
Fig. 2. (a) Size distribution of magnetic nanoparticles with anti-CAP. The magneticnanoparticle coated with anti-CAP is schematically shown in the inset. (b) Schemeof magnetic nanoparticle coated with anti-CAP which is labeled with fluorescentmarker FITC. (c) Green spot emitted from the fluorescent marker FITC observedwith a fluorescent microscope. This green spot moves downward in (d) undermagnetic attraction. The observed movement of the green spot in (c and d)evidences that the fluorescent marker FITC is on magnetic nanoparticle via thelinker of anti-CAP. (For interpretation of the references to colour in this figurelegend, the reader is referred to the web version of this article.)
ing. The results are shown in Fig. 2a. It was found that the meanvalue and the standard deviation for the diameter of magneticnanoparticles are 63 nm and 3.5 nm, respectively.
To investigate the immobilization of anti-CAP on magneticnanoparticles, goat-anti-rat-FITC antibody (Abcam), which is con-jugated to anti-CAP, was used as a fluorescent bio-marker to labelanti-CAP, as schematically shown in Fig. 2b. After the incubation ofcomplexes formed with the fluorescent bio-marker and the anti-CAP on magnetic nanoparticles, unbound fluorescent bio-markerswere removed from the reagent via magnetic separation.
The fluorescent marker FITC absorbs 495-nm in-wavelengthultraviolet light and then emits 528-nm in-wavelength green light.Thus, the positions of magnetic nanoparticles coated with anti-CAPcan be identified with green spot lights by using a fluorescentmicroscope.
Using a fluorescent microscope with a green-light band-passfilter (500–540 nm in wavelength), Fig. 2c shows the images of di-luted reagent mixed with goat-anti-rat-FITC antibody. As indicatedwith the arrow, a green spot was observed. This green spot wasgenerated with FITC. In order to verify that the goat-anti-rat-FITCantibody is on a magnetic nanoparticle, a magnet was put besidethe diluted reagent to move magnetic nanoparticles via magneticattraction. Therefore, the green spot observed in Fig. 2d wasexpected to move if goat-anti-rat-FITC was bound to the magneticnanoparticle. The movement of the green spot toward the magnetwas then observed, as illustrated in the image in Fig. 2d. The greenspot in Fig. 2d is located at a different position from that in Fig. 2c.This result shows that goat-anti-rat-FITC is on the magneticnanoparticle. Since the linking between goat-anti-rat-FITC andmagnetic nanoparticle is anti-CAP, the results shown in Fig. 2cand d reveal that the co-coating of anti-CAP is on magneticnanoparticles.
3.2. Standard curve of IMR on CAP
Various amounts of CAP (C1863-25G, Sigma) were added intopH-7.4 PBS solutions to achieve standard CAP solutions for variousconcentrations of 0.1–30 ng/g. Through triple IMR tests for stan-dard CAP solutions of each concentration, the IMR signal as a func-tion of CAP concentration /CAP was experimentally obtained andplotted with round points in Fig. 3. It was found in Fig. 3 that the
0.1 1.0 10.0 100.0φCAP (ng/g)
0.50
1.00
1.50
2.00
2.50
IMR
(%)
Noise level
CAP in PBS
CAP in Shrimp/wExtraction processes
Fig. 3. IMR signal as a function of the concentration of chloramphenicol (CAP) /CAP
added in PBS solution (round points) and shrimp (cross points). The solid line andthe dashed line are obtained by fitting round-point and cross-point data to Eq. (2),respectively.
(a)
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
IMR
(%
)
1 2 3 4 5 6Solution No.
p = 0.74p = 0.23
p = 0.38p = 0.42
p = 0.25
(b)
0.0 10.0 20.0 30.0φCAP via LS/MS/MS (ng/g)
0.0
10.0
20.0
30.0
φ CA
P v
ia IM
R (
ng/g
)
R2 = 0.98
Fig. 4. (a) IMR signals for solutions consisted of various contaminants forinterference tests. The constitutions of the six solutions are listed in Table 1. (b)Correlation between the detected CAP concentrations via LC/MS/MS and IMR in theblind tests for nine samples listed in Table 2.
1024 S.Y. Yang et al. / Food Chemistry 131 (2012) 1021–1025
IMR signal reduced with the decreasing CAP concentration. TheIMR signal approaches the noise level (�0.82% as guided with thedashed line) of the magnetic immunoassay analyzer as /CAP
becomes lower than 0.1 ng/g.According to published results, the concentration dependent
IMR signal follows logistic function. Hence, the experimental datashown in Fig. 3 are fitted to logistic function:
IMR ð%Þ ¼ A� B
1þ /CAP/o
� �b þ B ð2Þ
where A, B, b, and /o are fitting parameters. Through fitting theexperimental data to the above Eq. (2), A, B, b, and /o were foundto be 0.81, 4.54, 0.61, and 60.4, respectively. The fitted logistic curvewas plotted with the solid line in Fig. 3. The correlation coefficientR2 between the data points and the fitted logistic function is 0.98,which shows a fact of high correlation. This evidences that theIMR–/CAP curve belongs to the logistic function.
3.3. Interference test of IMR on CAP
Since CAP is just one of veterinary drug residues in foods, it isnecessary to analyze not only CAP but also other kinds of veteri-nary drug residues. This motivates the investigation of interferenceon analyzing CAP from other kinds of veterinary drug residues withIMR. Some typical veterinary residues such as malachite green(MG), leucomalachite green (LMG), 3-amine-2-oxazolidinone(AOZ), sulfamerazine (SMR), and oxytetracycline (OTC) are usedas interference materials for CAP analysis via IMR. Six solutions,which were featured in Table 1, were prepared for the interferencetests. It must be noted that these six solutions have the same con-centration for CAP. Except solution 1, interference materials of10 ng/g were added into each solution, respectively. Then, theIMR signals for these six solutions were detected using reagentwith anti-CAP, as shown in Fig. 4a. It was found that there is no sig-nificant difference among IMR signals of these six solutions. Aquantitative analysis of the consistence in IMR signals betweensolutions 2–6 and solution 1 was done through T-test statistic anal-ysis. A quantity, p value, was calculated for the consistence in IMRsignals between solutions 2–6 and solution 1, as labeled in Fig. 4a.The p values between solution 1 and solution 2 to solution 6 are0.74, 0.23, 0.38, 0.42 and 0.25, respectively. According to T-teststatistics, the two groups showing p value higher than 0.05 arereferred as identical. Therefore, solutions 2–6, which have interfer-ence materials, are identical to solution 1. This implies that theanalysis of CAP via IMR is independent of existence from otherkinds of veterinary drug residues.
3.4. IMR on extracted CAP from shrimp
To demonstrate the validity of the processes for extracting CAPfrom shrimp, a given amount of CAP was added while pulping theshrimp. In the experiment, 100-ng CAP was added into 10-g ofshrimp to have a test sample with CAP of 10 ng/g in concentration.
Table 1Constitutions of six solutions used for interference testson analyzing chloramphenicol (CAP) via immunomag-netic reduction.
Solution No. Constitution
1 5 ng/g CAP2 5 ng/g CAP and 10 ng/g MG3 5 ng/g CAP and 10 ng/g LMG4 5 ng/g CAP and 10 ng/g AOZ5 5 ng/g CAP and 10 ng/g SMR6 5 ng/g CAP and 10 ng/g OTC
Through the extraction processes developed in this work, the ex-tracted CAP solution was analyzed with IMR to find the concentra-tion of CAP in the solution. According to results of triple tests, theIMR signal of this extracted CAP solution was 1.70 ± 0.01%, whichresults in a CAP concentration of 8.8 ng/g, calculated from Eq. (2).This proves the validity to extract CAP from shrimp by using theprocesses. Furthermore, with the original concentration of10 ng/g, the extracting efficiency was 88%.
There was a difference in the measured IMR signal for10 ng/g CAP added in PBS solution and in shrimp. However, it isnecessary to extract CAP from shrimp. Hence, the standard curvefor CAP added in PBS solution shown in Fig. 3 is not adequate. Itwould be better to re-build a standard curve for CAP in shrimpto replace the solid line shown in Fig. 3. To do this, CAP solutionswith various concentrations were added in shrimp, followed byutilizing the extraction processes. The IMR signal as a function ofCAP concentration /CAP was investigated. The results were plottedwith cross points in Fig. 3. The cross points were then fitted into Eq.(2), which is guided with the dashed line in Fig. 3. As expected, alower IMR signal was observed. The lower IMR signal was attrib-uted to the non-100% extraction efficiency of CAP from shrimp asstated before.
3.5. Blind tests using IMR and LC/MS/MS
To examine the reliability of detecting CAP in shrimp using IMR,ten shrimp samples with added CAP of various concentrations
Table 2Concentrations of CAP added in shrimps, detected via LC/MS/MS and IMR,respectively.
Shrimp No. Added /CAP
(ng/g)/CAP via LC/MS/MS(ng/g)
/CAP via IMR(ng/g)
1 15 14.6 14.582 0.3 0.4 0.233 0 0.3 N/A4 20 20.6 17.365 0.2 0.3 N/A6 0 N/A N/A7 5 6.5 4.258 0.1 0.3 N/A9 25 27.1 20.8
10 0 N/A N/A
S.Y. Yang et al. / Food Chemistry 131 (2012) 1021–1025 1025
were prepared. Each sample was divided into two: one for IMR as-say and the other for LC/MS/MS assay. It should be noted that thetechnicians operating IMR and LC/MS/MS did not know the CAPconcentrations in these nine samples, as they were conductingblind tests. For IMR assay, the dashed line shown in Fig. 3 was usedas the standard curve. The results for CAP concentrations in thesenine samples detected with LC/MS/MS and IMR were shown inTable 2. It was found that the coefficient of correlation R2 in theCAP concentrations detected with LC/MS/MS and IMR was 0.98,as shown in Fig. 4b.
4. Conclusions
A method for analyzing the concentration of chloramphenicol(CAP) by using immunomagnetic reduction (IMR) was demon-strated. The reagent was mainly consisted of magnetic nanoparti-cles coated with antibody against CAP (anti-CAP), which was ableto bind with CAP to generate the reduction in the alternative-current magnetic susceptibility of the reagent. According to theexperimental results, the method was featured with a low detec-tion limit for analyzing CAP and low interference from other kindsof veterinary drug residues. Additionally, the convenient extractionprocesses for CAP from shrimp were developed and validated. Thewhole platform, including convenient extraction processes andIMR analysis, showed potential for practical applications.
Acknowledgements
This work has been supported by the National Science Councilof Taiwan under Grant Numbers 98-2112-M-003-003, 98-2323-B-003-001-CC2, and by the Department of Health under GrantNumbers DOH98-TD-N-111-008, and NSC98-2752-M-002-016-PAE, and by the Ministry of Economic Affairs of Taiwan underGrant Numbers 1Z970688 (SBIR), 1Z0990415 (SBIR), andS09800226-203 (JAID).
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