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Food Control 35 (2014) 26e32

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Food Control

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A simple quantum dot-based fluoroimmunoassay method for selectivecapturing and rapid detection of Salmonella Enteritidis on eggs

Beibei Wang, Xi Huang, Meihu Ma, Qing Shi, Zhaoxia Cai*

National R&D Center for Egg Processing, Food Science and Technology College, Huazhong Agricultural University, 1 Shizishan Street, Wuhan, Hubei 430070,China

a r t i c l e i n f o

Article history:Received 16 April 2013Received in revised form9 June 2013Accepted 15 June 2013

Keywords:Quantum dotFluoroimmunoassaySalmonella EnteritidisEggs

* Corresponding author. Tel.: þ86 027 87283177.E-mail address: caizhaoxia@mail.hzau.edu.cn (Z. C

0956-7135/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.foodcont.2013.06.025

a b s t r a c t

The aim of this paper was to demonstrate a rapid and selective fluorescence-linked immunoassaymethod based on quantum dots as the fluorescent marker for the detection of Salmonella Enteritidis oneggshell. Highly-fluorescent and water-soluble CdTe quantum dots were prepared by using thioglycolicacid and 1-thioglycerol as ligands and were then conjugated with anti-Salmonella antibodies. As a resultof specific interaction, Salmonella Enteritidis were specifically captured by bioconjugated CdTe quantumdots which led to the detection of a fluorescent signal. The bacterial cell images were obtained usingfluorescence microscopy. Under optimal conditions, the quenched fluorescence intensity increased lin-early with the log total count of Salmonella Enteritidis ranging from 3 � 102 to 3 � 107 CFU/mL within 1e2 h. The low detection limit was 1 � 102 CFU/mL without sample enrichment. This method wassatisfactorily applied to the analysis of egg samples, which was demonstrated as a simple scheme forquick and selective detection of Salmonella Enteritidis on eggshell.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Food contamination with Salmonella is an important publichealth concern in the whole world. Among all the Salmonella se-rotypes, Salmonella Enteritidis is identified as the main cause ofhuman salmonellosis infection, thus infecting thousands of peopleevery year (González-Escalona, Brown, & Zhang, 2012; Liu et al.,2012). The incidence of S. Enteritidis infections and the number ofrelated outbreaks have increased dramatically since 1970s. Besides,the accumulating evidences have indicated that eggs are the mostimportant source of human S. Enteritidis infection (Braden, 2006;Reu et al., 2006; Zhang, Zheng & Xu, 2011). Intensive epidemio-logic and laboratory investigations have identified that fresh eggs(obtained from the chicken farm directly without any process) canbe contaminated easily with S. Enteritidis in any contaminatedenvironment, such as the nest box, the hatchery environment orthe hatchery truck. In addition, eggs can be contaminated bypenetration through the eggshell from the colonized gut (Messens,Grijspeerdt, & Herman, 2005). S. Enteritidis can slower their ownmetabolism under the disadvantageous conditions on the dryeggshell surface. Therefore, S. Enteritidis can survive and grow onthe eggshell in the absence of faecal contamination and will survive

ai).

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for a longer time even at a low temperature (Gantois et al., 2009;Messens, Grijspeerdt, & Herman, 2006; Radkowski, 2002). Aboveall, one of the effective measures to reduce the infection from S.Enteritidis on the eggshell is to develop cleaned eggs which havebeen processed by washing, disinfecting under ultraviolet irradia-tion, drying, coating preservative, and packing before selling (Xiao,Zhang, Chi, Feng, & Xiao, 2013). However, for most ordinary eggs, arapid, simple, highly sensitive and selective detection method of S.Enteritidis on eggshell is in urgent need.

The conventional identification approaches include culture andcolony counting (Allen, Edberg, & Reasoner, 2004), enzyme linkedimmunosorbent assay (Cheung & Kam, 2012), polymerase chainreaction (Martin, Garriga, & Aymerich, 2012; Salinas, Garrido,Ganga, Veliz, & Martınez, 2009), immunological techniques (Van,Ieven, Pattyn, Van, & Laga, 2001) and fluorescence-based assays(Fu, Huang, & Liu, 2009). Although these methods are error-proofand powerful, most of them are labour-intensive, complex andtime-consuming for a complete analysis (Sanvicens Pastells,Pascual, & Marco, 2009). Therefore, the highly-sensitive and rapiddetection methods are expected.

Nowadays, fluorescence analysis, which is developed for rapid,technically-simple and efficient detection of the bacterial countfrom originals or pre-enriching samples without any expensiveapparatus, is of great importance (Luo et al., 2009). Quantum dots(QDs), a family of nanosized particles, comprised of a few thousandatoms with typical sizes of 1e10 nm in radius. They have been

Fig. 1. Schematic diagram of the coupling strategy.

Fig. 2. The entire immunoassay procedure for detection of S. Enteritidis.

B. Wang et al. / Food Control 35 (2014) 26e32 27

widely used in the fields of chemistry and biomedical science as afluorescence marker (Chan & Nie, 1998). Compared to conventionalorganic fluorophores, QDs have many advantages such as broad-band excitation, narrow and symmetric emission spectra, highsignal-to-noise ratios, considerably greater resistance to quenchingand photobleaching (Duong & Rhee, 2007; Xue, Pan, Xie, Wang, &Zhang, 2009). Thus, water-soluble QDs are the ideal fluorescencemarkers in the detection of total bacterial count (Agasti et al., 2010;Bae et al., 2010; Jackeray et al., 2011; Sanvicens et al., 2011; Tully,Hearty, Leonard, & O’Kennedy, 2006).

In this study, we prepared the monodispersive, homogeneous,highly-fluorescent and water-soluble CdTe QDs, and then conju-gated prepared CdTe QDs with anti-S. Enteritidis antibodies. It wasa novel method to use this bioconjugated CdTe QDs as fluorescentlabel for rapid, technically-simple and efficient detection of S.Enteritidis. The fluorescence intensity was linear with the bacterialcount in the range of 3 � 102e3 � 107 CFU/mL and the low detec-tion limit was 1 � 102 CFU/mL in 1e2 h. The method was alsoapplied to the direct detection of the S. Enteritidis count in real eggsamples to prove its feasibility and practicability. The resultsdemonstrated that the application of bioconjugated CdTe QDs asfluorescent probes in detecting S. Enteritidis count on eggshell wasrapid, selective and feasible.

2. Experimental

2.1. Materials and reagents

All reagents were of analytical grade and used without priorpurification. Doubly-deionized water (DDW) was used throughoutthis work. NaBH4, CdCl2$2.5H2O, NaOH, thioglycolic acid (TGA), N-hydroxysuccinimide (NHS), Nutrient broth medium (NB), nutritionagar medium and salts studied (Naþ, Kþ), glucose, lactose wereacquired from Guoyao Company (China). Glycine, cysteine, gluta-thione, L-serine, tyrosine, albumin, lysozyme, BSA were purchasedfrom Biosharp Company (China). Te powder was acquired fromTianjin Delan Fine Chemical Reagent Company (China). 1-Thioglycerol (TG) was acquired from Aladdin Chemical ReagentCompany Ltd. (China). 1-ethyl-3-[3-dimethylaminopropyl] carbo-diimide hydrochloride (EDC) was acquired from Aladdin, Inc.(China). Rabbit anti-Salmonella antibody (3e4 mg/mL) was pur-chased from Gene Tex, Inc. (USA).

Phosphate buffered solution (PBS) with different pH values wereprepared by mixing 1/15 mol/L Na2HPO4 and 1/15 mol/L NaH2PO4according to certain proportions.

The egg samples used in the experiment were obtained from alocal chicken farm and the supermarket in China. Three types ofeggs were used in this experiment: fresh eggs from a local chickenfarm, ordinary eggs from the supermarket and the cleaned eggsfrom the supermarket. The cleaned eggs had been processed bywashing, disinfecting under ultraviolet irradiation, drying, coatingpreservative, and packing (Xiao et al., 2013). Each sample wasprepared by using three eggs during the experiment. The eggsamples were put into sterile plastic bags respectively. Their egg-shells were rubbed with 200 mL sterile DDW for 1e2 min to detachthe bacteria. The wash solutions from eggshell were collected forfuture detection. For the stability of the experiment, each samplewas set in three parallel.

2.2. Synthesis and characterization of CdTe QDs

Highly-fluorescent and water-soluble CdTe QDs were synthe-sized in aqueous solution based on a previous public method withminor modification (Yang & Gao, 2005). Briefly, the oxygen-freeNaHTe solutions were prepared by stirring a mixture of 36.4 mg

NaBH4, 26.7 mg Te powder and 1.5 mL DDW at room temperatureuntil black Te powder disappeared. The freshly-prepared oxygen-free NaHTe solutions were reacted with a mixture of 160 mL0.026mol/L CdCl2 and 140 mL TG, 27.5 mL TGA (pH 11.2 adjustedwith1 mol/L NaOH) at 97 �C for 1 h.

The QDs concentration c (¼ A/(εL)) was measured according toCarion, Mahler, Pons, and Dubertret (2007). In this equation, A wasthe absorption of the QDs sample measured at 350 nm, L was theoptical path (usually the dimension of the cuvette used for themeasurement) and ε (M�1 cm�1) ¼ (1.438 � 1026) a3 was theextinction coefficient at 350nm (awas theQD radius in centimetres).

The quantumyield (QY) of CdTe QDswas calculated according toliterature (Brouwer, 2011).

Synthesized hydrophilic CdTeQDswere characterized by varioustechniques.

Absorption spectra were obtained using a UV-1800 pharmaspectrometer (Shanghai, China). Fluorescence intensity and emis-sion spectra were recorded with the excitation wavelength fixed at330 nm using an RF-5301 spectrophotometer (Hitachi, Japan). Allthe spectra were recorded with DDW as blank. The image of CdTeQDs were examined by an JEM-2100F high-resolution transmissionelectron microscopy (JEOL, Japan) with an acceleration voltage of200 kV. The aqueous solutions of QDs were dropped on an 800-mesh carbon-coated copper grid and dried at room temperaturefor HRTEM sample preparation.

The X-ray powder diffraction spectra (XRD) patterns were ob-tained on a D/max-2500 X-ray diffractometer with Cu Ka radiation(Rigaku, Japan). XRD samples were prepared as follows. The QDssolution was concentrated to about one tenth using a rotaryevaporator and then cold ethanol was added until turbidityoccurred. The precipitate and supernatant were separated bycentrifugation. To prepare the final powder sample, the ethanolprecipitated and re-dissolved samples were dried overnight undervacuum at 60 �C.

2.3. Bioconjugation of CdTe QDs with anti-S. Enteritidis antibodies

Fig.1 showed the coupling process between CdTe QDs and anti-S.Enteritidis antibodies. Thewater-soluble CdTeQDswere activated byusing carbodiimide chemistry for conjugationwith anti-S.Enteritidisantibodies. Particularly,1mL (QY 36%, about 1.02�10�6mol) of QDsstock solution was put into the mixed solutions containing 1 mL ofPBS (0.01 mol/L, pH 7.2) and 100 mL (4 mg/mL) of EDC. The mixture

Fig. 5. XRD images of CdTe QDs.Fig. 3. UVevis absorbance (a) and fluorescence spectra (b) of water-soluble CdTe QDs.

B. Wang et al. / Food Control 35 (2014) 26e3228

was under gentle stirring at room temperature for 5 min and it tookabout 15e20 min to fully activate free carboxylic acid groups on theQDs (Zhao et al., 2009). Then, 100 mL (0.15 mg/mL) of sulfo-NHS wasadded to themixed solutions and themixturewas stirred for another20min. Thereafter, anti-S. Enteritidis antibodieswere added into thesystem and reacted for at least 2 h in constant temperature incu-bator. In this step, CdTe QDs and anti-S. Enteritidis antibodies wereconjugated through strong covalent bonds. The final bioconjugatedCdTe QDs were collected and kept at 0e4 �C overnight. Conjugationprocess was optimized by varying reaction temperature and time aswell as themole ratios of CdTeQDs and anti-S. Enteritidis antibodies.

2.4. Bacterial inoculate preparation and surface plating methods

Stock cultures of S. Enteritidis were obtained from our laboratoryculture preservation. Cultureswere grown for 18e24h at 37 �C inNB.Serial 10-fold dilutions were made in sterile PBS. The number of S.Enteritidis was determined by surface plating technology using0.1 mL proper dilutions onto nutrition agar medium. After the in-cubation at 37 �C for 24 h, colonies on the plates were counted todetermine the number of visible cells in the cultures in terms ofcolony forming units per millilitre (CFU/mL). For safety consider-ations, all of the bacterial sampleswere inactivated in an autoclave at121 �C for 15min, except for some special tests andcharacterizations.

Fig. 4. HRTEM image of CdTe QDs.

2.5. Detection of S. Enteritidis by immunoassay procedure

The basic immunoassay procedures for detection of the S.Enteritidis were displayed in Fig. 2. After the S. Enteritidis cultureswere centrifuged, poured out of the supernatant and suspended theprecipitated S. Enteritidis in 1 mL of axenic PBS. The process wasrepeated twice to remove the NB. Then, the serial 10-fold of S.Enteritidis culture dilutions were made in sterile PBS. For quanti-tative determination, 200 mL of the bioconjugated CdTe QDswas putinto certain amounts of S. Enteritidis culture (3�102 to 3�107 CFU/mL, respectively) and theywere kept at 37 �C for 40min. In this step,bioconjugated CdTe QDswere used as fluorescence labels to capturetarget bacterial cells through the immuno-recognition betweenantibodies and S. Enteritidis. 1mL DDWwas treated, using the sameprocedure, as a control. After the reactions, these mixtures werecentrifuged at 8000 rpm for 10min to precipitate the S. Enteritidisebioconjugated CdTe QDs. The resulting supernates were used tomonitor the change of emission fluorescence intensity by spectro-fluorometer. The calibration graph set up in the study was used toquantify of the concentration of S. Enteritidis in the sample. Theexcitation wavelength was selected at 330 nm. The slit widths ofboth excitation and emission were 5.0 nm.

Fig. 6. Fluorescence spectra of CdTe QDs before (a) and after (b) bioconjugation.

Fig. 7. Changes in fluorescence spectra of the bioconjugated CdTe QDs in the presenceof different concentrations of Salmonella Enteritidis. Concentration of S. Enteritidis (a)to (k) (CFU/mL): 3 � 102, 1.8 � 103, 6 � 103, 1.2 � 104, 1.8 � 104, 6 � 104, 1.8 � 105,1.8 � 106, 2.4 � 106, 1.2 � 107, 3 � 107; Concentration of CdTe QDs: 1.02 � 10�6 mol/L,lex ¼ 330 nm; pH 7.2.

Fig. 8. Fluorescence microscopy image of S. Enteritidis cells attached to bioconjugatedCdTe QDs with emission wavelength of 512 nm (green). (For interpretation of thereferences to colour in this figure legend, the reader is referred to the web version ofthis article.)

Fig. 9. (A) Effect of immunologic process reaction time. (B) Effect of temperature in the absenof (a) and in the presence of S. Enteritidis (b). Concentration of CdTe QDs: 1.02 � 10�6 mol

B. Wang et al. / Food Control 35 (2014) 26e32 29

2.6. Fluorescence microscopy

A fluorescence microscope (Olympus, BX71), equipped with adigital CCD camera (Olympus DP70) and a broadband mercurylamp (OSRAM HBO 100 W) with an ultraviolet excitation filter, wasused to capture images of the samples on the glass slides.

3. Results and discussion

3.1. Synthesis and characterization of CdTe QDs

Fig. 3 shows UVevis absorbance (a) and fluorescence spectra (b)of CdTe QDs. The absorption spectrum indicates that CdTe QDs has awider range of absorption with the absorption peak at 460 nm.Their broad absorption spectra allow for the efficient excitation atany wavelength with a single light source. The emission spectrumshows characteristically narrow and symmetric emission peak at512 nm. The CdTe QDs provide sufficient spectral resolution forquantitative detection of the fluorescence intensity with 40 nm ofFWHM (full width at half maximum) and high QY (36%).

3.2. HRTEM image of CdTe QDs

Fig. 4depicts theHRTEMimageof theCdTeQDs.Theshapeof theseQDs was spherical, crystalline, sufficiently-monodisperse and well-separated, with the average size about 2e3 nm. It also shows thatlattice planes are extended across the entire particle, which confirmsa well-crystallized structure of the CdTe QDs (He & Gu, 2006).

3.3. XRD spectra of the CdTe QDs

The XRD resulted in Fig. 5 confirms the formation of cubicstructure of CdTe QDs. The peaks observed at 2q are 24.4�, 41.6�,46.9�, corresponding to the (111), (220) and (311) crystalline planesof cubic CdTe (Huang, Liu, Han, Mi, & Xu, 2012). The considerablybroad width of the diffraction peaks indicates that the CdTe QDshave a nano-size distribution which is consistent with the image ofHRTEM.

3.4. Optimum conditions for detection

Fig. 6 displays the fluorescence emission spectra of CdTe QDsbefore (a) and after (b) being bioconjugated with anti-S. Enteritidisantibodies. After being bioconjugated, the fluorescence intensityincreases and shifts slightly towards higher wavelength because ofthe surrounding organic layer (anti-S. Enteritidis antibodies) ofCdTe QDs (Goldman et al., 2004).

ce of (a) and in the presence of Salmonella Enteritidis (b). (C) Effect of pH in the absence/L, lex ¼ 330 nm; pH 7.2.

Table 1Interference of coexisting substances.

Coexistingsubstances

Concentration(10�6 mol/L)

Change of fluorescenceintensity (%)

Naþ 10000 þ3.9Kþ 10000 þ4.8Glycine 400 �2.6Glucose 3000 �3.2Lactose 4000 þ2.7Cysteine 300 þ2.3Glutathione 100 þ5.0L-serine 200 þ4.9Tyrosine 200 þ4.3Albumin 3 �4.9Lysozyme 0.3 �4.8BSA 0.2 þ3.9

Table 2Comparison of the linear ranges and LODs of several selected fluorimetric methodsfor determination of S. Enteritidis.

Reagent Linear range(CFU/mL)

LOD(CFU/mL)

Reference

Quantum dots coatedwith streptavidin

1.0 � 103e1.0 � 107 1.0 � 103 Yang and Li (2005)

Q dot� 705 streptavidinconjugate

104e107 104 Yang and Li (2006)

CdSe/ZnS QDs NDa 10 CFU/g Wang et al., (2012)CdTe quantum dots 3.0 � 102e3.0 � 107 1.0 � 102 This work

a ND: not mentioned.

B. Wang et al. / Food Control 35 (2014) 26e3230

The coupling process conditions were optimized by varying theconcentration of anti-Salmonella antibodies, reaction temperatureand pH. Fig. S1 shows the effect of the concentration of anti-S.Enteritidis antibodies on bioconjugation system. Maximum fluo-rescence intensity occurs at the concentration of 7.5 mg/mL. Whenconcentration is higher than 7.5 mg/mL, the limited QDs moleculescannot occupy all binding sites of anti-S. Enteritidis antibodiescoexisting in the system. When the concentration is lower than7.5 mg/mL, the fluorescence intensity may decease because of theself-quenching effect of excessiveQDs. Fig. S2 shows the influence ofreaction temperatures on bioconjugation. As is shown, the reactionwas completed within 120 min at 37 �C. Fig. S3 shows the effect ofpHon the bioconjugated process. FromFig. S3,we can see thatwhenpH is 7.2, the fluorescence intensity reaches a maximum andtherefore the pH of 7.2 is consequently selected for further studies.

3.5. Detection of S. Enteritidis using bioconjugated CdTe QDs

3.5.1. The interaction of bioconjugated CdTe QDs with S. EnteritidisChanges offluorescence spectra of the bioconjugated CdTe QDs in

the presence of different concentrations of S. Enteritidis are pre-sented in Fig. 7. The fluorescence signals decrease rapidly with therising S. Enteritidis concentrations (from 3� 102 to 3� 107 CFU/mL).Thus, themore S.Enteritidis bacterial cells are in the sample, themore

Fig. 10. Linear relationships of the changes in fluorescence intensity versus the log-transformed cell population during the detection of S. Enteritidis, at populationsranging from 3 � 102e3 � 107 CFU/mL. Concentration of CdTe QDs: 1.02 � 10�6 mol/L,lex ¼ 330 nsm; pH 7.2.

bioconjugated CdTe QDs could bind. The total detection time, fromadding a sample solution to obtaining the final result, is less than 2 h.

3.5.2. Fluorescence microscopyFluorescence microscopy image of S. Enteritidis cells attached to

bioconjugated CdTe QDs with emission wavelengths of 512 nm(green) are showed in Fig. 8. The fluorescence signals of the resultswere definitively confirm that the S. Enteritidis were captured bybioconjugated CdTe QDs.

3.5.3. Optimum conditions for detectionIn order to perfect the fluoroimmunoassay method of S. Enter-

itidis, the test conditions were improved by studying the effect ofvarious factors such as reaction time, temperature and pH. Theresults were shown in Fig. 9. Fig. 9A show that the immunologicprocess reaction can be completed within 40 min. As the temper-ature is an important factor in the fluorescencemeasure, it has beeninvestigated how the fluorescence intensity changes at varyingtemperatures during detection, and the results are shown in Fig. 9B.Themaximumvalue of fluorescence intensity are reachedwhen thesystem has been detected at 0 �C (changes of the fluorescence in-tensity in the presence and absence of S. Enteritidis). Therefore, 0 �Cwas adopted in this work. Fig. 9C shows the effect of pH on thesystem. It is found that the optimum pH ranges are from 7.2 to 7.5.When pH is lower than 7.2 or greater than 7.5, the fluorescenceintensity is comparatively lower. The reason may be explained asfollows: in low pH value, the fluorescence intensity decreases as apossible result of the deconstruction of the Cd2þeTGA complexes’annulus due to the protonation of the surface-binding thiolates.When pH value is too high, the fluorescence intensity decreasespartly because of the electrostatic repulsion between electronega-tive S. Enteritidis and bioconjugated CdTe QDs (Yu et al., 2007). Inthis work, the pH of 7.2 is selected.

3.5.4. Analytical performanceThe effect of some coexisting substances such as Naþ, Kþ,

glycine, glucose, lactose, cysteine, glutathione, L-serine, tyrosine,albumin, lysozyme, BSA were investigated. The experimental re-sults are listed in Table 1. It indicates that most of the coexistingsubstances, such as metal ions, amino acid and some proteins, havelittle interference on the determination of S. Enteritidis within the

Table 3Results for the determination of S. Enteritidis in synthetic sample.

Syntheticsample

Main interferences(10�6 mol/L)

Change of fluorescenceintensity (%)

1 Naþ 10000, Kþ 10000, glutathione 50,BSA 0.1, glucose 1500, lactose 1000

�4

2 Naþ 10000, cysteine 150, glucose 1500,glutathione 50, BSA 0.1

�1.3

3 Naþ 10000, Kþ 10000, glutathione 50,BSA 0.1

10.6

Table 4Results for the determination of S. Enteritidis in egg samples.

The variety of eggs Sample Plate countmethod (CFU/mL)

This method(CFU/mL)

Eggs from chicken farm Sample 1 103 1.85 � 103

Sample 2 8 � 102 8.29 � 102

Sample 3 NDa NDEggs from supermarket Sample 1 ND ND

Sample 2 3 � 102 3.98 � 102

Sample 3 ND NDCleaned eggs from

supermarketSample 1 ND NDSample 2 ND NDSample 3 ND ND

a ND: not detected.

B. Wang et al. / Food Control 35 (2014) 26e32 31

permission of 5.0% relative error, which demonstrates a good ca-pacity of resisting disturbance in this method.

According to the above procedures, the calibration curve for thedetermination of S. Enteritidis was constructed under the optimalconditions. Fig. 7 shows the emission spectra of the bioconjugatedCdTe QDs in the presence of various concentrations of S. Enteritidis.The fluorescence intensity is significantly quenched with the in-crease of S. Enteritidis. The linear relationships between thechanges of fluorescence intensity and the log-transformed S.Enteritidis population are shown in Fig. 10. The linear ranges for S.Enteritidis are 3 � 102e3 � 107 CFU/mL and the linear regressionequation isDF¼ 80.80C� 156.6(C¼ log CFU/mL) (DF¼ F0� F), witha correlation coefficient of 0.9958. The limit of detection (LOD) isdefined by the equation LOD ¼ 3S0/K, where S0 is the standarddeviation of blank measurements (n ¼ 10) and K is the slope ofcalibration graph. Here LOD is 1 � 102 CFU/mL.

For comparative purpose, the linear ranges and LODs of severalselected fluorimetric methods for S. Enteritidis detection using QDsare summarized in Table 2. From the table, we can see that thedetections have a relatively low LOD, however, the cost and thecomplexity of experiment are increased in these methodsemploying the step of bead-free or magnetic beads isolation (Wang,Wu, Fan, & Mustapha, 2012; Yang & Li, 2005). The simple methodproposed in the study has a wider linear range and higher sensi-tivity without enrichment and isolation.

According to the interference of foreign substances, Table 3 givesthe results of analysis for the three synthetic samples. As is shown,the values found for the three synthetic samples are identical withthe expected values. Therefore, the method proposed has beenproven to be sensitive, simple and reliable.

To further prove feasibility and practicability of this method, itwas applied to detect the total bacterial count in egg samples. All ofthe eggs needed to be washed to collect the solutions from eggshellas samples. Table 4 shows the test results of egg samples in ourmethod and the conventional plate count method respectively. Asis shown, the results of our method are consistent with the con-ventional plate count method. Thus, the use of bioconjugated CdTeQDs as fluorescence labels in immunoassays method is applicableto directly detect the S. Enteritidis. S. Enteritidis cannot be detectedon the surface of cleaned eggs in this study. The reason may be thatafter being washed and disinfected under ultraviolet irradiation,nearly no S. Enteritidis can survive on the eggshells. What is more,the film coating on the eggshells is another effective measurementto reduce bacterial contamination (Leleu et al., 2011; Xie et al.,2002). Therefore, the development and spread of cleaned eggswill be of great significance in the future.

4. Conclusions

In conclusion, this study have synthesized high-quality CdTeQDs as fluorescence labels for rapid, sensitive detection of the total

S. Enteritidis count in immunoassays method. The fluorescenceintensity decreases with the increase of total count of S. Enteritidisand a linear relationship can be established. The results of deter-mination for the total count of S. Enteritidis in egg samples areidentical with the conventional plate count method. The studydemonstrates that the application of CdTe QDs as immunoassaysfluorescent probes is feasible to detect the total bacteria count.

Acknowledgements

This research was supported by the National Basic ResearchProgram of China (No. 31101290).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.foodcont.2013.06.025.

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