Chemiluminescence Resonance Energy Transfer CompetitiveImmunoassay Employing Hapten-Functionalized Quantum Dots forthe Detection of SulfamethazineMingfang Ma,† Kai Wen,‡ Ross C. Beier,§ Sergei A. Eremin,⊥ Chenglong Li,† Suxia Zhang,†,‡

Jianzhong Shen,†,‡ and Zhanhui Wang*,†,‡

†Beijing Advanced Innovation Center for Food Nutrition and Human Health, College of Veterinary Medicine, China AgriculturalUniversity, No. 2 Yuanmingyuan West Road, Beijing 100193, China‡Beijing Laboratory for Food Quality and Safety and Beijing Key Laboratory of Detection Technology for Animal-Derived FoodSafety, No. 2 Yuanmingyuan West Road, Beijing 100193, China§Food and Feed Safety Research Unit, Southern Plains Agricultural Research Center, Agricultural Research Service, United StatesDepartment of Agriculture, 2881 F&B Road, College Station, Texas 77845, United States⊥Faculty of Chemistry, M. V. Lomonosov Moscow State University, Leninsky Gory, Moscow 119992, Russia

*S Supporting Information

ABSTRACT: We describe a new strategy for usingchemiluminescence resonance energy transfer (CRET) byemploying hapten-functionalized quantum dots (QDs) in acompetitive immunoassay for detection of sulfamethazine(SMZ). Core/multishell QDs were synthesized and modifiedwith phospholipid-PEG. The modified QDs were function-alized with the hapten 4-(4-aminophenyl-sulfonamido)-butanoic acid. The CRET-based immunoassay exhibited alimit of detection for SMZ of 9 pg mL−1, which is >4 orders ofmagnitude better than a homogeneous fluorescence polar-ization immunoassay and is 2 orders of magnitude better thana heterogeneous enzyme-linked immunosorbent assay. This strategy represents a simple, reliable, and universal approach fordetection of chemical contaminants.

KEYWORDS: quantum dots, chemiluminescence resonance energy transfer, immunoassay, hapten functionalization,chemical contaminant

Contamination of food by chemical hazards is a worldwidepublic health concern and is a leading cause of problems

in international trade.1 Consequently, Regulatory Authoritiesand the food/feed industries have large budgets for monitoringand controlling the safety of food products. These chemicalcontaminants include a wide range of compounds likeveterinary drugs, pesticides, mycotoxins, hormones, packagingcomponents, and drugs of abuse.2 Thus, antibody-based assays,i.e., the immunoassay, is one of the most important techniquesfor detecting these contaminants in a variety of food samplesbecause of the high sensitivity, specificity, reproducibility, andanalysis speed, and are referred to as enzyme-linkedimmunosorbent assays (ELISAs), lateral flow immunoassays(LFAs) and electrochemical immunoassays.3 However, mostimmunoassays are heterogeneous and require several separationsteps with strict washing procedures, which are time-consumingand tedious. Therefore, it is necessary to develop a simple andeffective homogeneous immunoassay for the detection ofchemical contaminants. Unlike the heterogeneous immuno-assay that requires binding at a surface, the homogeneousimmunoassay binds in solution, drastically reducing the

distance that antigens must diffuse to reach the antibodies.4

Combined with the removal of washing steps, the total assaytime is reduced, making the homogeneous immunoassay moresuitable for rapid detection applications. Even with theseadvantages, only a few homogeneous immunoassays forchemical contaminants have been demonstrated including afluorescence polarization immunoassay (FPIA),5 a fluorescenceresonance energy transfer (FRET)6 immunoassay and aluminescence oxygen-channeling immunoassay (LOCI).7

Our group has developed a number of FPIAs during the pastdecade for chemical contaminants, including antibiotics andmycotoxins, and these studies have shown the advantages ofFPIA including decreased assay time, reduced labor, and theability to be fully automated.5,8 However, the overall sensitivityof the FPIA is usually lower compared with the conventionalELISA. The sensitivity may not be acceptable when an analysis

Received: April 8, 2016Accepted: June 30, 2016Published: June 30, 2016

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© 2016 American Chemical Society 17745 DOI: 10.1021/acsami.6b04171ACS Appl. Mater. Interfaces 2016, 8, 17745−17750

needs to be run at trace levels of an analyte. Moreover, limitedapproaches are available to enhance the FPIA sensitivity in largepart because it lacks a signal amplification step during thedetection procedure. Another type of homogeneous immuno-assay is becoming popular and relies on resonance energytransfer (RET) that occurs between a donor and an acceptor inaqueous solution.9 Several studies have developed the FRETimmunoassay for detection of low-molecular-weight analytessuch as explosives, hormones, vitamins, and pesticides.10

Compared with the well-established FRET system, thechemiluminescence RET, or CRET, exhibits superior perform-ance that occurs by the oxidation of a luminescent substratethat excites the acceptor without the need for an external lightsource.11,12 Hence, it can reduce the autofluorescence back-ground and the fluorescence bleaching. Therefore, CRET is anattractive light-measuring scheme useful in bioassays. In theconventional CRET, the fluorophores acting as energyacceptors usually have a small Stokes shift, which result inpoor spectral separation between the acceptor emission and thedonor emission and low energy-transfer efficiency.11 Semi-conductor nanocrystals, known as quantum dots (QDs), haveemerged as a unique new class of fluorescence materials overthe past decade. QDs are capable of high quantum yield,improved sensitivity, and high photostability. They have size-tunable emission wavelengths, and have paved the way fornumerous studies including imaging, sensing and targetingbiomolecules.3,13 Thus, QDs are now rapidly replacingtraditional fluorophores in almost all fluorescence-basedapplications.14 However, it is only recently that QDs havebeen applied to CRET systems. Ren et al.15 were the first todemonstrate efficient CRET between luminol as the energydonor and CdTe core QDs as the acceptor, based on animmuno-interaction of BSA-QDs and anti-BSA-HRP in theluminol-H2O2 chemiluminescence reaction. Since the work ofRen et al.,15 the QD-based CRET has now been applied to thedetection of small molecules (e.g., H2O2, adenosine triphos-phate and glucose), DNA, and proteins.14,16 In theseapplications, core or core/shell QDs were used and theywere usually functionalized with macromolecules, such asproteins, antibodies, and an aptamer.17 A key approach toimprove the detection sensitivity of the CRET-based bioassay isto enhance the CRET efficiency. Although the QD-CRETefficiency is dependent on several factors, the quantum yield ofthe acceptor QD is the crucial factor to the CRET efficiency;18

that is, a high quantum yield of the QDs results in a high CRETefficiency. Studies have demonstrated that the passivation ofcore QDs containing multishells strongly increases theluminescence of the QDs up to 35−50% quantum yield.18

In spite of the promising possibility offered by CRET, themethod has so far only been focused on in vivo imaging studiesrather than on the development of bioanalysis. There are noreports of CRET-based immunoassays for small moleculedetection, where hapten-functionalized QDs are used to achievehigh sensitivity in a competitive immunoassay format. In thisstudy, we developed a CRET-based competitive immunoassayusing hapten-functionalized core/multishell QDs as an energyacceptor for detection of the chemical contaminant, sulfame-thazine (SMZ). SMZ was used as a model because it is themost frequently used sulfonamide (SA) in veterinary clinicsthroughout the world (Figure S1). The performance of theCRET-based immunoassay showed significant superioritycompared with the common homogeneous FPIA andheterogeneous ELISA.

The assembly of the CRET-based competitive immunoassayfor SMZ is shown in Scheme 1. In Scheme 1A, the amphiphilic

polymer modified core/multishell QDs were functionalizedwith the hapten molecule (BS), and the catalyst, HRP, waslinked to the specific antibody (mAb4D11) resulting in mAb-HRP. When the mAb-HRP specifically binds to the BS-QDsand the donor luminol was added to the system including H2O2and enhancer p-iodophenol, CRET can occur resulting in theincrease of fluorescence intensity of BS-QDs. In Scheme 1B,when the analyte SMZ is involved in the CRET system, it canblock the CRET process by inhibiting the binding between BS-QDs and mAb-HRP. With an increase in SMZ concentration, itwill result in a decrease in fluorescence intensity of the BS-QDs,and the reverse is also true.The highly luminescent core/multishell QDs were prepared

in organic solvents according to our previous report (see theSupporting Information).19 For bioanalytical applications, aligand layer is necessary to transfer the hydrophobic QDs to anaqueous solution. Since the original hydrophobic ligandsremain on the QD surface, coating the QDs with amphiphilicmolecules is preferred to better maintain the initial bright-ness.20,21 Moreover, the employment of amphiphilic polymers,rather than single molecules or di- or triblock systems, ispreferable because polymer chains contain multiple hydro-phobic cells which can result in strong interactions with theinitial organic coating. Some amphiphilic polymers have beenused to modify QDs; however, most of these polymers werehomemade through complicated processes and are notcommercially available.22−24 In this study, we used acommercially available and low-cost amphiphilic polymer(DSPE-PEG-NH2) to modify the hydrophobic QDs by areverse-phase evaporation technique (see the SupportingInformation).19 The size and morphology of the obtainedhydrophilic QDs were studied under a high-resolutiontransmission electron microscope (HRTEM). The HRTEMimage of a single modified QD particle revealed highcrystallinity with continuous lattice fringes throughout thewhole particle (Figure 1A) and the blurry appearance at the rimof this particle may be due to the molecular ligands. TheFourier transform of the HRTEM image (dFT(100) = 3.50 Å) inFigure 1A inset was in good agreement with that of the QDparticles prepared by Xie et al.25 Figure 1B, C demonstrates theuniform spherical shape of the modified QDs having about a6.5 nm diameter and being fairly dispersed. In addition to

Scheme 1. (A) Schematic Illustration of the CRET Processbetween the Donor, Luminol, And the Acceptor, QDs, Basedon the Immunoreaction between mAb-HRP and BS-QDs;and (B) Schematic Illustration of the CRET-BasedCompetitive Immunoassay for SMZ

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HRTEM analysis, the hydrodynamic diameter of 20.7 nm wasalso studied by dynamic light scattering (DLS) and shown inFigure 1D. The polydispersity index (PDI) of the QDs sizedistribution analyzed by DLS was less than 0.1, indicating thatthe size distribution of the QDs is narrow and shows goodmonodispersity.We are the first to describe here hapten functionalized QDs

for the development of a CRET-based competitive immuno-assay. The carboxylic group of hapten BS was conjugated to theamino group of the QDs using carbodiimide. Prior to the BS-QDs being used in the CRET-based immunoassay, they werecharacterized with X-ray photoelectron spectra (XPS), UV−visspectra, fluorescence spectra, HRTEM and immunochemicalactivity. Figure 2A shows the XPS spectra of the BS-QDs, QDs,and BS. High-resolution XPS can provide nanoscale sensitivityfor analysis of QD surface ligand exchange or conjugation.However, the limitation of XPS in this study rests on the factthat the polymer-modified QDs contain all elements of thehapten BS, resulting in all peaks being integrated. Therefore,qualitative confirmation of the conjugation of BS to the QDswas uncertain, which is similar to the report by Zou et al.26 Thehighest binding energy peak intensity obtained from the BS-QDs was in agreement with the incorporation of BS moleculesto the QDs. Then, the UV−vis spectrum and the fluorescencespectrum for the QDs and BS-QDs at the same concentrationwere investigated as shown in Figure 2B. It can be seen that theUV−vis spectrum (left in Figure 2B) of the QDs was slightlyshifted after BS incorporation. The fluorescence spectra (rightin Figure 2B) was not shifted but was slightly reducedsuggesting it may be due to conjugation of BS to the surface ofthe QDs resulting in slight fluorescence quenching. This resultsuggests that the BS molecule does not have much adverseimpact on the fluorescence characteristics of the QDs.Subsequently, the immunochemical active BS-QDs were furtherstudied in the CRET-based immunoassay. Two negativecontrol experiments with an anti-ERM mAb-HRP (instead of

the anti-SAs mAb-HRP) and the unconjugated QDs withoutBS and a positive control experiment with addition ofconjugated SMZ were performed under identical conditions.Figure 2C shows the fluorescence intensity of the assayobtained under different conditions: BS-QDs+anti-SAs mAb-HRP, BS-QDs+anti-SAs mAb-HRP+SMZ, BS-QDs+anti-ERMmAb-HRP, and QDs+anti-SAs mAb-HRP. It is not surprisingthat the highest fluorescence intensity was obtained during theincubation of the BS-QDs and anti-SAs mAb-HRP. A slightlysmaller fluorescence intensity was observed in the positivecontrol experiment, which was attributed to the inhibition ofthe SMZ binding between BS-QDs and the anti-SAs mAb-HRP, resulting in blocking energy transfer. Much lessfluorescence intensity was obtained from the two negativecontrol experiments since there was no specific bindingoccurring between these reagents. These results indicated thatthe hapten-functionalized QDs provided immunochemicalactivity signifying that the hapten-QDs had a small enoughdiameter, which guarantee the occurrence of CRET. Inaddition, the HRTEM in Figure 2D shows the mondispersityof the BS-QDs following functionalization of the QDs with BS.The number of BS molecules per QD was calculated bycomparing the concentration of BS and the concentration ofQDs in the conjugation solution according to a previousreport.26 On average, 16 BS molecules were linked to a singleQD. The SDS-PAGE study of the QDs and BS-QDs alsoindicated that the exact mass and charge of the BS-QDs did notsignificantly increase in the BS functionalized QDs compared tothe QDs (see Figure S2). This is in agreement with thecharacterization by XPS and fluorescence spectra.To develop a highly sensitive CRET-based immunoassay for

SMZ, we first optimized the buffer system and workingconcentration of the BS-QDs. As illustrated in Figure 3A, theluminol chemiluminescence intensity at 425 nm in phosphatebuffered saline (PBS, 10 mM, pH 7.4) (green line) was higherthan the intensity in borate buffer (BB, 50 mM, pH 7.4) (blackline) when the BS-QDs were absent in the CRET system underthe same conditions. However, the highest fluorescence

Figure 1. (A) HRTEM images (at 2 nm) of QDs and the Inset showsthe Fast-Fourier transform of the HRTEM image (dFT(100) = 3.50 Å),(B) HRTEM images (at 20 nm) of QDs, (C) HRTEM images (at 50nm) of QDs, and (D) hydrodynamic size distribution of the QDs byDLS.

Figure 2. (A) XPS measurement of the BS-QDs, QDs, and BS; (B)typical UV−vis (left, 425 nm) and fluorescence (right, 610 nm)spectra of the QD and BS-QDs; (C) fluorescence intensity of the assayunder different conditions, and (D) HRTEM images (at 20 nm) of theBS-QDs.

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intensity at 610 nm (inset 3A) was observed when the BS-QDswere added to PBS (blue line) rather than being added to theborate buffer (red line), indicating that in this study, PBS wasthe more optimal buffer of choice for the chemiluminesencereaction and energy transfer. Unlike sandwich-type immuno-assays, the highest sensitivity can be obtained in a competitiveimmunoassay by having the lowest possible probe (BS-QDs)concentrations theoretically required. Generally, the workingconcentration of the BS-QDs should be kept close to theminimum detectable concentration for the instrument in use,which will allow a reliable detection of the BS-QDs but doesnot affect the competition. In this study, the concentra-tion of BS-QDs was evaluated and the results are shown in

Figure 3B, demonstrating that a 1:40 dilution of the originalBS-QDs (1 μM) could induce a measurable fluorescence signaland that concentration was selected for use in the assay. Basedon the above optimized conditions, a calibration curve (FigureS6) containing various concentrations of SMZ was determinedby the immunoassay, and the limit of detection (LOD) wasobserved at 9 pg mL−1 (3 times the standard deviation of theblank, 3σ) and the analytical range was 0.01−50 ng mL−1. TheLOD of the assay was far below the maximum residue limit ofSMZ (100 ng mL−1). Because the anti-SAs mAb4D11 hadbroad-specific binding ability,27 three other SA analogs werealso used to evaluate the assay specificity. The calibration curvesfor these SAs are shown in Figure S6B−D, providing cross-reactivities of 100, 539.98, 47.27, and 84.58% for SMZ,sulfadimethoxine (SDM), sulfaquinoxaline (SQX) and sulfa-methoxazole (SMX), respectively.To demonstrate the CRET-based immunoassay for SMZ in

real samples, we determined the precision (% CV) and theaccuracy (% recovery) values of the assay for SMZ atconcentrations of 50, 100, and 200 μg L−1 in milk. Theaccuracy values as expressed in % recovery ranged from 64.7 to110.7%. The % CV values were less than 10.6% at all

concentrations, indicating that the assay may be a potentialmethod for analyzing milk (Table 1).

There are many reports that compare different antibody-based analytical methods for the detection of chemicalcontaminants such as for microcystins28 and sulfonamides.29

The analytical performance comparison among immunoassaysare objective and meaningful only when the same pair ofantibody−antigens are employed in the different immunoassayformats. Both the antibody and antigen reagents significantlycontribute to the sensitivity and specificity of the immunoassay.In this study, we developed an FPIA for SMZ based on apreviously reported ELISA for the detection of SMZ, and weused the same antibody (anti-SAs mAb4D11) and antigen (BS-AMF in the FPIA and BS-BSA in the ELISA).30 The principle,procedure and results of the FPIA for SMZ can be found inSupporting Information section 5, Figures S5 and S6 and TableS1. The detailed parameters from the three immunoassays areshown in Table 1 and the standard curves are shown in Figure3C. The calculated LODs of the FPIA and ELISA for SMZwere 12.1 ng mL−1 and 0.151 ng mL−1 in buffer, respectively(Table 1). The sensitivity of the CRET-based immunoassay forSMZ is more than 4 orders of magnitude better than the FPIAand was even more than 2 orders of magnitude better than theELISA, indicating that the CRET-based immunoassay incor-porating nanomaterials can significantly improve assaysensitivity. The specificities of the three immunoassays wereevaluated using three SA analogues other than SMZ and areshown in Figure 3D. The cross-reactivities of the immunoassaysfor the four SAs studied showed similar tendencies, indicatingthat the recognition profiles of the antibody do not vary as theimmunoassay format was changed.We are the first group to describe a homologous CRET-

based competitive immunoassay for the detection of SMZincorporating BS-functionalized core/multishell QDs. Thismethod is an excellent substitute for a FRET-based immuno-assay because it eliminates the necessity for an externalexcitation source. The CRET-based immunoassay exhibited aLOD of 9 pg mL−1, an assay time of 10 min and required nosample preparation. Therefore, the CRET-based immunoassayis a suitable method to be used as a fast, simple, sensitivescreening tool for chemical contaminants. By comparison withFPIA and ELISA, the CRET-based immunoassay offerssignificant advantages. The CRET-based immunoassay devel-oped here can easily be extended to other chemicalcontaminants by simply changing the targets of interest.Thus, the CRET-based immunoassay represents a generalstrategy for food safety residue analysis.

Figure 3. (A) Chemiluminance intensity and fluorescence intensity(inset) in the CRET-based immunoassay under different conditions;(B) fluorescence intensity with different concentrations of the BS-QDs; (C) standard curves for SMZ by CRET, ELISA, and FPIA,where S/S0 is the normalized response variable; and (D) cross-reactivities of the ELISA, FPIA, and CRET for SMZ, SMX, SDM, andSQX.

Table 1. Comparisons of Analytical Performance of CRETwith ELISA and FPIAa

CRET ELISAb FPIAc

LOD (ng mL−1)d 0.009 0.151 12.1IC50 (ng mL−1) 0.2 ± 0.03 3.084 ± 0.5 98.7 ± 4.3detection range (ngmL−1)

0.01−50 0.6−15.7 32−145

assay time (min) <10 >180 <20accuracy (% recovery) 64.7−110.7 83.4−102.5 75.4−125.3precision (% CV) <10.6 <16.0 <16.3aUsing SMZ as a model analyte. bFrom ref 30. cSee SupportingInformation. dThe data were obtained from the calibration curves inbuffer.

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■ ASSOCIATED CONTENT

*S Supporting InformationThe Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acsami.6b04171.

Experimental details including materials, synthesis andfunctionalization of multishell QDs, preparation andcharacterization of mAb-HRP and tracer BS-AMF,development of CRET and FPIA, Figures S1−S7, andTable S1 (PDF)

■ AUTHOR INFORMATION

Corresponding Author*E-mail: wangzhanhui@cau.edu.cn. Fax: 861062731032.

Author ContributionsAll authors have given approval to the final version of themanuscript.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors thank the National Natural Science Foundation ofChina (Grant 31372475) for financial support.

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