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1 Detection of Epstein Barr virus infection in cancerby using highly specific nanoprobe based on dBSAcapped CdTe quantum dots Yilin Li,abLihong Jing,bKe Ding,bJing Gao,aZhi Peng,aYanyan Li,aLin Shen*aand Mingyuan Gao*bBy using denatured bovine serum albumin (dBSA) molecules as multidentate ligands, surface-functionalizedCdTe quantum dots (QDs) were synthesized and further evaluated for preparingfluorescent probes capableof detecting Epstein Barr virus infection. The optical studies revealed that the dBSA coating effectivelyimproved the optical stability of the resulting QDs The interaction between CdTe@dBSAand the membrane of carcinoma cells further suggested that the nonspecific adsorption of CdTe QDsstabilized by thioglycolic acid (TGA) was significantly eliminated upon the dBSA coating. Streptavidin wascovalently conjugated to CdTe@dBSA to endow the QDs with specific binding affinity in detecting theserum level of anti-Epstein Barr virus (EBV) capsid antigen IgA (VCA-IgA) in nasopharyngeal carcinomapatients for early screening and diagnosing EBV-associated IntroductionQuantum dots (QDs) have been attracting considerable atten-tion owing to their unique electronic and optical 3As the spectral features of QDs in comparison with that ofconventional dyes is characterized by narrow, symmetric, andparticle size-dependent uorescence, as well as a broad excita-tion range and excellent robustness against photobleaching,QDs are ideal on many counts for bioassay and 7 However, due to the strong surface defect-sensitive uores-cence, the emission intensity of QDs only coated with a smallthiol ligand, such as thioglycolic acid (TGA), mercaptopropionicacid (MPA)

2 , and so forth, is readily reduced in 10 Most importantly, the surface of the QDs isalways prone to adsorbing biomolecules nonspeci cally,11 13which sharply decreases the speci city of the QDs-basedmolecular probes, and thereby limits the bioapplication of strategies for surface modi cation on QDs have beendeveloped to overcome these 20 However, the majorstrategies, such as the encapsulation of QDs with silica,21 23organic polymers,24 27or amphiphilic polymers15,17,18,20haveconcentrated on passivating the surface defects of QDs therebyimproving their uorescence ,28 Limited approacheshave been developed to suppress the nonspeci c interactionbetween QDs and biomolecules. Polyethylene glycol (PEG)remains the most commonly used material to modify the surfaceof nanoparticles for suppressing their non-speci c adsorptionwith ,14,29 Owing to the antifouling property ofPEG, the binding speci city of PEG-coated particle probes can beeffectively improved for immuno uorescence ,30 Even forin vivoapplications, PEG-coated QDs also exhibit reducednonspeci c accumulation in the reticuloendothelial tissues,which is bene cial for increasing the accumulation of QDs atregion of addition, multidentate PEG ligands havealso been developed for effectively passivating the 19,31 However, the preparation of multidentate PEGligands requires sophisticated synthetic procedures.

3 Moreover,the functional groups, typical at the end of the PEG molecules,limit the number of bioligands to conjugate to PEGylated QDs,and thereby their potential in bioassays and bovine serum albumin (dBSA) has recently beenused to manipulate the surface chemistry of ,33 Preparedby chemically reducing disul de bonds in BSA, dBSA contains37 thiol groups per monomer, thereby affording them greatpotential as excellent multidentate ligands for from that, BSA is also a well-known blocking reagent toreduce nonspeci c adsorption in immunoassays similar 36It is therefore reasonable to expect that dBSA as mul-tidentate ligands can not only passivate surface defects andimprove the optical properties of QDs, but also suppress theaKey Laboratory of Carcinogenesis and Translational Research (Ministry of Education),Department of GI Oncology, Peking University Cancer Hospital and Institute, FuchengRoad 52, Haidian District, Beijing 100142, China.

4 E-mail: +86-10-88196561bInstitute of Chemistry, the Chinese Academy of Sciences, Bei Yi Jie 2, Zhong Guan Cun,100190 Beijing, China. E-mail: Tel: +86-10-82613214 Electronic supplementary information (ESI) available: The uorescenceintensity and peak position of CdTe@dBSA with different dBSA : QD ratios, andthe optical stability of CdTe@dBSA by optimal dBSA : QD ratio in 1 PBS arerespectively shown in Fig. S1 and S2; the uorescent stability of CdTe@dBSA streptavidin bioconjugate are shown in Fig. S3. See this:RSCAdv.,2014,4, 22545 Received 15th March 2014 Accepted 1st May 2014 DOI: journal is The Royal Society of Chemistry 2014 RSCAdv.,2014,4, 22545 22550 |22545 RSC AdvancesPAPER nonspeci c interaction of QDs with biomolecules. Moreover,compared with PEG polymer, dBSA contains different func-tional groups such as amino and carboxyl groups along thepolymer backbone, which provides more reactive sites forconjugation with bioligands to achieve QD-based probes forboth bioassay and this rationale, dBSA was chosen to encapsulateCdTe QDs stabilized by TGA in this study.

5 The optical propertiesof the resulting CdTe@dBSA were systematically studied. Theinteraction between CdTe@dBSA and carcinoma cells was thenstudied to investigate the resistance of dBSA coating to non-speci c binding. Furthermore, the CdTe@dBSA streptavidinconjugate was synthesized and used as a molecular probe todetect Epstein Barr virus (EBV) infection in nasopharyngealcarcinoma (NPC) patients. EBV, as a member of the herpes virusfamily, has been implicated in the occurrence of variouscancers, such as NPC, Burkitt's lymphoma, gastric carcinoma,and so er infected by EBV, patients always exhibitelevated level of anti-EBV antibodies in sera, particularly theanti-EBV capsid antigen IgA (VCA-IgA).37 39 Therefore, detectionof the serum level of VCA-IgA has served as an effective strategyfor early screening and diagnosis of EBV-associated ,39In this paper, the application of the CdTe@dBSA streptavidinconjugate in detecting the serum level of VCA-IgA in NPCpatients was also demonstrated through indirect immuno uo-rescence Materials and ChemicalsAluminum telluride (Al2Te3, ), cadmium perchloratehydrate (Cd(ClO4)2$6H2O, ), thioglycolic acid (TGA, 97+%),1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, +%),N-hydroxysulfo-succinimide sodium salt (Sulfo-NHS, ),were all purchased from Sigma-Aldrich.

6 Agarose (BiowestAgarose) was purchased from Gene Tech. Co., Ltd., Shanghai,China. Streptavidin was purchased from Promega Biotech Co.,Ltd. Bovine serum albumin (BSA, Amresco, 99%), acrylamide(AM, Amresco, 99+%),N,N0-methylenebis(acrylamide) (MBA,Amresco, 99%), ammonium persulfate (APS, Amresco, 95%),N,N,N0,N0-tetramethylethylenediamin e (TEMED, Sigma, 99%),tris(hydroxymethyl)aminomethane (Tris, Amresco, ),glycine (Gly, Amresco, ) were distributed by BiodeeBiotechnology Co., Ltd., Beijing, China. Goat anti-humanimmunoglobulin A antibody (antiIgA Ab) was gi ed from theNational Institute for Viral Disease Control and Prevention,China CDC. Other chemicals were all purchased by BeijingChemical Factory, Beijing, China. All the chemicals mentionedabove were used as Synthesis of CdTe QDsAqueous CdTe QDs stabilized by TGA were synthesized, by thereaction between Cd2+and H2Te, according to the methodreported ,40 42 However, in the present study theinitial pH of the precursor solution was set to 12 and the ratio ofCd : HSR was adjusted from 1 : to 1 : The resulting CdTeQDs with emission wavelength centered at 610 nm was used inthe following Preparation of CdTe@dBSA QDsdBSA was prepared according to previous ,33,43 Brie y,NaBH4was added to BSA solution and reacted under stirring for1 h.

7 Then, the resultant solution was further heated to 60 80 Cuntil no more gas (H2) was generated, in order to decomposethe excess NaBH4. Under these conditions, most of the disul debonds of BSA were reduced to thiol groups. The resultant dBSAsolution was dialyzed against PBS buffer, and stored at 4 C forfurther preparing CdTe@dBSA QDs, the dBSA was mixed withTGA-coated CdTe QDs in 1 PBS to exchange the TGAligands. This ligand exchange reaction typically lasted for 24 hat room temperature. The preparation of dBSA andCdTe@dBSA was carried out under anaerobic condition. A erthat, excess dBSA was removed by centrifugal ultra ltrationusing Amicon 100 KD centrifugal ultra ltration units (Milli-pore). The nally prepared CdTe@dBSA was characterizedusing 10% sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE). Preparation of CdTe@dBSA streptavidin bioconjugateBefore the conjugation reaction, the dBSA coating on CdTe QDswas succinylated by using succinic anhydride to convert theprimary amine groups to carboxyl groups according to theprotocol reported by Kuoet resulting succinylatedCdTe@dBSA was activated in 1 PBS buffer containing EDC andsulfo-NHS (CdTe@dBSA : EDC : sulfo-NHS 1 : 1000 : 2500) for15 min under gentle stirring.

8 Then streptavidin was introduced(CdTe@dBSA : streptavidin 1 : 2 or 1 : 1) under gentle mixingfor 4 h in 1 PBS. The resulting CdTe@dBSA streptavidin bio-conjugate was centrifuged by using Amicon 100 KD centrifugalultra ltration units to remove unreacted streptavidin, and thenstored at 4 C for 24 h before further Detection of VCA-IgA in NPC patientsThe indirect immuno uorescence method was adopted todetect VCA-IgA in the sera of NPC patients and healthydonors. In detail, the EBV-activated human lymphoma Rajicells (gi s from National Institute for Viral Disease Controland Prevention, China CDC) were xed on glass slides. Thesera diluted by a factor of 5 were introduced for incubatingwith the cells at 37 C for 30 min. A er that, the biotinylatedantiIgA Ab was added a er the cells were washed 3 times byusing 1 PBS. The following incubation at 37 C was allowedfor another 30 min. A er being washed 3 times with 1 PBS,the cell slide was subjected to incubation with theCdTe@dBSA streptavidin bioconjugate at room temperaturefor 30 min.

9 The cells nally obtained were imaged underOlympus IX71 inverted uorescence microscope a er beingwashed with 1 |RSCAdv.,2014,4, 22545 22550 This journal is The Royal Society of Chemistry 2014 RSC AdvancesPaper3. Results and dBSA-coated CdTe QDsThe absorption and uorescence spectra of the as-preparedCdTe QDs stabilized by TGA, denoted as CdTe@TGA, are shownin Fig. 1a. The room temperature uorescence quantum yield(QY) was estimated to be 61% according to ,45 Representative transmission electron microscope(TEM) and high-resolution TEM (HRTEM) images of theCdTe@TGA QDs are shown in Fig. 1b. The average size of theQDs is around nm according to the particle size histogramshown in Fig. con rm the formation of the CdTe@dBSA QDs by theligand exchange reaction, 10% SDS-PAGE was carried out andthe results are shown in Fig. 2a and b. The luminescent resultsreveal that the CdTe@dBSA (Lane 2) migrates far more slowlythan CdTe@TGA QDs (Lane 1), implying the effective coating ofdBSA on CdTe QDs.

10 Further silver staining results as presentedin Fig. 2b show that both dBSA (Lane 3) and CdTe@dBSA arestained owing to the existence of dBSA. According to previousstudies,32the three bands of dBSA showing according to thesilver staining results can be attributed to dBSA monomer,dBSA dBSA dimer, and globulins, respectively. Since the rsttwo bands of CdTe@dBSA exhibited by both uorescence andsilver staining match perfectly, it can be concluded that CdTeQDs coated by the monomer and dimer of dBSA were success-fully uorescence studies suggest that the dBSA coatingsigni cantly increases the uorescence intensity of the under-lying CdTe core, as shown in Fig. 2c. In contrast, suchenhancement effect was not presented upon simple mixing ofBSA with CdTe@TGA QDs. On the contrary, the uorescenceintensity of the CdTe@TGA QDs is slightly decreased. Theseresults support that dBSA can effectively passivate the CdTe coreby eliminating of the surface addition, dBSAmolecule contains a large number of thiol groups, which is veryhelpful for eliminating the Cd-related achieve high performance CdTe@dBSA for immuno uo-rescenceassay,theeffectofthedBSA-to-Q Dsratioon uorescencewas studied.