Journal of Fluorescence

, Volume 23, Issue 4, pp 649–657 | Cite as

A Novel Homogeneous Time-Resolved Fluoroimmunoassay for Carcinoembryonic Antigen Based on Water-Soluble Quantum Dots

  • Zhen-Hua Chen
  • Ying-Song Wu
  • Mei-Jun Chen
  • Jing-Yuan Hou
  • Zhi-Qi Ren
  • Da Sun
  • Tian-Cai Liu


Quantum dots are not widely used in clinical diagnosis. However, the homogeneous time-resolved fluorescence assay possesses many advantages over current methods for the detection of carcinoembryonic antigen (CEA), a primary marker for many cancers and diseases. Therefore, a novel luminescent terbium chelates- (LTCs) and quantum dots-based homogeneous time-resolved fluorescence assay was developed to detect CEA. Glutathione-capped quantum dots (QDs) were prepared from oil-soluble QDs with a 565 nm emission peak. Conjugates (QDs-6 F11) were prepared with QDs and anti-CEA monoclonal antibody. LTCs were prepared and conjugates (LTCs-S001) were prepared with another anti-CEA monoclonal antibody. The fluorescence lifetime of QDs was optimized for sequential analysis. The Förster distance (R0) was calculated as 61.9 Å based on the overlap of the spectra of QDs-6 F11 and LTCs-S001. Using a double-antibody sandwich approach, the above antibody conjugates were used as energy acceptor and donor, respectively. The signals from QDs were collected in time-resolved mode and analyzed for the detection of CEA. The results show that the QDs were suitable for time-resolved fluoroassays. The spatial distance of the donor-acceptor pair was calculated to be 61.9 Å. The signals from QDs were proportional to CEA concentration. The standard curve was LogY = 2.75566 + 0.94457 LogX (R = 0.998) using the fluorescence counts (Y) of QDs and the concentrations of CEA (X). The calculated sensitivity was 0.4 ng/mL. The results indicate that water-soluble QDs are suitable for the homogenous immunoassay. This work has expanded future applications of QDs in homogeneous clinical bioassays. Furthermore, a QDs-based homogeneous multiplex immunoassay will be investigated as a biomarker for infectious diseases in future research.


Water-soluble quantum dots Homogeneous immunoassay FRET Terbium chelates Carcinoembryonic antigen 

List of Abbreviations

6 F11

Anti-CEA monoclonal antibody




Bis(sulfosuccinimidyl) suberate sodium salt


Carcinoembryonic antigen




Anhydrous dimethyl sulfoxide


Diethylene triamine pentacetic acid


Diethylenetriaminepentaacetic acid dianhydride




(N-(3-dimethyllaminopropyl) carbodiimide hydrochloride


Ethylenediaminetetraacetic acid


Förster resonance energy transfer


Full-width at half maximum


Glutathione reduced


Homogeneous time-resolved fluoroassays


Luminescent terbium chelates


Monoclonal antibody


2-[N-morpholino]ethanesulfonic acid


Oil-soluble quantum dots


Phosphate buffered saline


Quantum dots


Quantum yield


Anti-CEA monoclonal antibody


Standard deviation


N-hydroxysulfosuccinimide sodium salt


Tri-n-octylphosphine oxide


Time-resolved Förster resonance transfer




Water-soluble quantum dots



The work was supported by the National Natural Science Foundation of China (Grant No. 30901382, 81271931), the Natural Science Foundation of Guangdong Province (No. S2012010009547), the New Teacher for Doctoral Fund of Ministry of Education of China (Grant No. 20094433120008), Special Funds for College and University Talents by Guangdong Province (2009) and Scientific Research Foundation of Introducing Talents of Southern Medical University(2009).

Competing interests



The National Natural Science Foundation of China (Grant No. 30901382, 81271931), the New Teacher for Doctoral Fund of Ministry of Education of China (Grant No. 20094433120008), the Natural Science Foundation of Guangdong Province (No. S2012010009547), Special Funds for College and University Talents by Guangdong Province (2009), and the Scientific Research Foundation of Introducing Talents of Southern Medical University(2009).

Ethical approval

The Ethical Committee of Science and Technology Department of Southern Medical University approved this study (REC number: 20121058B).


Tian-Cai Liu.


Mei-Jun Chen, Jing-Yuan Hou and Da Sun contributed to experimental work. Tian-Cai Liu and Ying-Song Wu researched literature and conceived the study. Mei-Jun Chen and Zhi-Qi Ren were involved in protocol development, gaining ethical approval, and data analysis. Mei-Jun Chen wrote the first draft of the manuscript. All authors reviewed and edited the manuscript and approved the final version of the manuscript.


  1. 1.
    Gold P, Freedman SO (1965) Demonstration of tumor-specific antigens in human colonic carcinomata by immunological tolerance and absorption techniques. J Exp Med 121:439–462PubMedCrossRefGoogle Scholar
  2. 2.
    Gold P, Freedman SO (1965) Specific carcinoembryonic antigens of the human digestive system. J Exp Med 122(3):467–481PubMedCrossRefGoogle Scholar
  3. 3.
    Tsai HL, Chang YT, Chu KS, Chen CF, Yeh YS, Ma CJ, Wu DC, Kuo CH, Chan HM, Sheen MC, Wang JY (2008) Carcinoembryonic antigen in monitoring of response to cetuximab plus FOLFIRI or FOLFOX-4 in patients with metastatic colorectal cancer. Int J Biol Markers 23(4):244–248PubMedGoogle Scholar
  4. 4.
    Duxbury MS, Ito H, Benoit E, Waseem T, Ashley SW, Whang EE (2004) A novel role for carcinoembryonic antigen-related cell adhesion molecule 6 as a determinant of gemcitabine chemoresistance in pancreatic adenocarcinoma cells. Cancer Res 64(11):3987–3993. doi: 10.1158/0008-5472.CAN-04-0424 PubMedCrossRefGoogle Scholar
  5. 5.
    Tanaka T, Huang J, Hirai S, Kuroki M, Watanabe N, Tomihara K, Kato K, Hamada H (2006) Carcinoembryonic antigen-targeted selective gene therapy for gastric cancer through FZ33 fiber-modified adenovirus vectors. Clin Cancer Res 12(12):3803–3813. doi: 10.1158/1078-0432.CCR-06-0024 PubMedCrossRefGoogle Scholar
  6. 6.
    Yoon SM, Shin KH, Kim JY, Seo SS, Park SY, Kang S, Cho KH (2007) The clinical values of squamous cell carcinoma antigen and carcinoembryonic antigen in patients with cervical cancer treated with concurrent chemoradiotherapy. Int J Gynecol Cancer 17(4):872–878. doi: 10.1111/j.1525-1438.2007.00878.x PubMedCrossRefGoogle Scholar
  7. 7.
    Chu TM, Reynoso G, Hansen HJ (1972) Demonstration of carcinoembryonic antigen in normal human plasma. Nature 238(5360):152–153PubMedCrossRefGoogle Scholar
  8. 8.
    Withofs M, Offner F, de Paepe P, Praet M (2000) Carcinoembryonic antigen elevation in agnogenic myeloid metaplasia. Br J Haematol 110(3):743–744PubMedCrossRefGoogle Scholar
  9. 9.
    Kodera Y, Isobe K, Yamauchi M, Satta T, Hasegawa T, Oikawa S, Kondoh K, Akiyama S, Itoh K, Nakashima I et al (1993) Expression of carcinoembryonic antigen (CEA) and nonspecific crossreacting antigen (NCA) in gastrointestinal cancer; the correlation with degree of differentiation. Br J Cancer 68(1):130–136PubMedCrossRefGoogle Scholar
  10. 10.
    Shousha S, Lyssiotis T, Godfrey VM, Scheuer PJ (1979) Carcinoembryonic antigen in breast-cancer tissue: a useful prognostic indicator. Br Med J 1(6166):777–779PubMedCrossRefGoogle Scholar
  11. 11.
    Ishiguro F, Fukui T, Mori S, Katayama T, Sakakura N, Hatooka S, Mitsudomi T (2010) Serum carcinoembryonic antigen level as a surrogate marker for the evaluation of tumor response to chemotherapy in nonsmall cell lung cancer. Ann Thorac Cardiovasc Surg 16(4):242–247PubMedGoogle Scholar
  12. 12.
    Hsu WH, Huang CS, Hsu HS, Huang WJ, Lee HC, Huang BS, Huang MH (2007) Preoperative serum carcinoembryonic antigen level is a prognostic factor in women with early non-small-cell lung cancer. Ann Thorac Surg 83(2):419–424. doi: 10.1016/j.athoracsur.2006.07.079 PubMedCrossRefGoogle Scholar
  13. 13.
    de Diego A, Compte L, Sanchis J, Enguidanos MJ, Marco V (1991) Usefulness of carcinoembryonic antigen determination in bronchoalveolar lavage fluid. A comparative study among patients with peripheral lung cancer, pneumonia, and healthy individuals. Chest 100(4):1060–1063PubMedCrossRefGoogle Scholar
  14. 14.
    van Nagell JR Jr, Donaldson ES, Wood EG, Goldenberg DM (1978) The clinical significance of carcinoembryonic antigen in the plasma and tumors of patients with gynecologic malignancies. Cancer 42(3 Suppl):1527–1532PubMedCrossRefGoogle Scholar
  15. 15.
    Shibata S, Raubitschek A, Leong L, Koczywas M, Williams L, Zhan J, Wong JY (2009) A phase I study of a combination of yttrium-90-labeled anti-carcinoembryonic antigen (CEA) antibody and gemcitabine in patients with CEA-producing advanced malignancies. Clin Cancer Res 15(8):2935–2941. doi: 10.1158/1078-0432.CCR-08-2213 PubMedCrossRefGoogle Scholar
  16. 16.
    Maziak W (2008) Carcinoembryonic antigen (CEA) levels in hookah smokers, cigarette smokers and non-smokers–a comment. J Pak Med Assoc 58(3):155PubMedGoogle Scholar
  17. 17.
    Stevens DP, Mackay IR (1973) Increased carcinoembryonic antigen in heavy cigarette smokers. Lancet 2(7840):1238–1239PubMedCrossRefGoogle Scholar
  18. 18.
    Urva SR, Yang VC, Balthasar JP (2009) Development and validation of an enzyme linked immunosorbent assay for the quantification of carcinoembryonic antigen in mouse plasma. J Immunoass Immunochem 30(4):418–427. doi: 10.1080/15321810903188227 CrossRefGoogle Scholar
  19. 19.
    Franchimont P, Debruche ML, Zangerlee PF, Proyard J (1973) Radioimmunoassay of the carcinoembryonic antigen. Ann Immunol (Paris) 124(4):619–630Google Scholar
  20. 20.
    Tsaltas G, Ford CH, Gallant M (1992) Demonstration of monoclonal anti-carcinoembryonic antigen (CEA) antibody internalization by electron microscopy, western blotting and radioimmunoassay. Anticancer Res 12(6B):2133–2142PubMedGoogle Scholar
  21. 21.
    Hou JY, Liu TC, Lin GF, Li ZX, Zou LP, Li M, Wu YS (2012) Development of an immunomagnetic bead-based time-resolved fluorescence immunoassay for rapid determination of levels of carcinoembryonic antigen in human serum. Anal Chim Acta 734(93):93–98. doi: 10.1016/j.aca.2012.04.044 PubMedCrossRefGoogle Scholar
  22. 22.
    Stockley RA, Shaw J, Whitfield AG, Whitehead TP, Clarke CA, Burnett D (1986) Effect of cigarette smoking, pulmonary inflammation, and lung disease on concentrations of carcinoembryonic antigen in serum and secretions. Thorax 41(1):17–24PubMedCrossRefGoogle Scholar
  23. 23.
    Sajid KM, Chaouachi K, Mahmood R (2008) Hookah smoking and cancer: carcinoembryonic antigen (CEA) levels in exclusive/ever hookah smokers. Harm Reduct J 5:19. doi: 10.1186/1477-7517-5-19 PubMedCrossRefGoogle Scholar
  24. 24.
    Dungchai W, Siangproh W, Lin JM, Chailapakul O, Lin S, Ying X (2007) Development of a sensitive micro-magnetic chemiluminescence enzyme immunoassay for the determination of carcinoembryonic antigen. Anal Bioanal Chem 387(6):1965–1971. doi: 10.1007/s00216-006-0899-y PubMedCrossRefGoogle Scholar
  25. 25.
    Yang X, Guo Y, Wang A (2010) Luminol/antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen. Anal Chim Acta 666(1–2):91–96. doi: 10.1016/j.aca.2010.03.059 PubMedCrossRefGoogle Scholar
  26. 26.
    Matsushita H, Xu J, Kuroki M, Kondo A, Inoue E, Teramura Y, Nozawa M, Senba T, Yamamoto T, Matsuoka Y (1996) Establishment and evaluation of a new chemiluminescent enzyme immunoassay for carcinoembryonic antigen adapted to the fully automated ACCESS system. Eur J Clin Chem Clin Biochem 34(10):829–835PubMedGoogle Scholar
  27. 27.
    Haggart R, Thorpe GH, Moseley SB, Kricka LJ, Whitehead TP (1986) An enhanced chemiluminescent enzyme immunoassay for serum carcinoembryonic antigen based on a modification of a commercial kit. J Biolumin Chemilumin 1(1):29–34. doi: 10.1002/bio.1170010106 PubMedCrossRefGoogle Scholar
  28. 28.
    Kricka LJ (1994) Selected strategies for improving sensitivity and reliability of immunoassays. Clin Chem 40(3):347–357PubMedGoogle Scholar
  29. 29.
    Dickson EF, Pollak A, Diamandis EP (1995) Ultrasensitive bioanalytical assays using time-resolved fluorescence detection. Pharmacol Ther 66(2):207–235PubMedCrossRefGoogle Scholar
  30. 30.
    Blomberg KR, Mukkala VM, Hakala HH, Makinen PH, Suonpaa MU, Hemmila IA (2011) A dissociative fluorescence enhancement technique for one-step time-resolved immunoassays. Anal Bioanal Chem 399(4):1677–1682. doi: 10.1007/s00216-010-4485-y PubMedCrossRefGoogle Scholar
  31. 31.
    Mathis G, Socquet F, Viguier M, Darbouret B (1997) Homogeneous immunoassays using rare earth cryptates and time resolved fluorescence: principles and specific advantages for tumor markers. Anticancer Res 17(4B):3011–3014PubMedGoogle Scholar
  32. 32.
    Selvin PR, Hearst JE (1994) Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. Proc Natl Acad Sci U S A 91(21):10024–10028PubMedCrossRefGoogle Scholar
  33. 33.
    Li M, Selvin PR (1997) Amine-reactive forms of a luminescent diethylenetriaminepentaacetic acid chelate of terbium and europium: attachment to DNA and energy transfer measurements. Bioconjug Chem 8(2):127–132. doi: 10.1021/bc960085m PubMedCrossRefGoogle Scholar
  34. 34.
    Leyris JP, Roux T, Trinquet E, Verdie P, Fehrentz JA, Oueslati N, Douzon S, Bourrier E, Lamarque L, Gagne D, Galleyrand JC, M'Kadmi C, Martinez J, Mary S, Baneres JL, Marie J (2011) Homogeneous time-resolved fluorescence-based assay to screen for ligands targeting the growth hormone secretagogue receptor type 1a. Anal Biochem 408(2):253–262. doi: 10.1016/j.ab.2010.09.030 PubMedCrossRefGoogle Scholar
  35. 35.
    Bruchez M Jr, Moronne M, Gin P, Weiss S, Alivisatos AP (1998) Semiconductor nanocrystals as fluorescent biological labels. Science 281(5385):2013–2016PubMedCrossRefGoogle Scholar
  36. 36.
    Chan WC, Nie S (1998) Quantum dot bioconjugates for ultrasensitive nonisotopic detection. Science 281(5385):2016–2018PubMedCrossRefGoogle Scholar
  37. 37.
    Gerion D, Pinaud F, Williams SC, Parak WJ, Zanchet D, Weiss S, Alivisatos AP (2001) Synthesis and properties of biocompatible water-soluble silica-coated CdSe/ZnS semiconductor quantum dots. J Phys Chem B 105(37):8861–8871. doi: 10.1021/jp0105488 CrossRefGoogle Scholar
  38. 38.
    Chan WC, Maxwell DJ, Gao X, Bailey RE, Han M, Nie S (2002) Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol 13(1):40–46PubMedCrossRefGoogle Scholar
  39. 39.
    Han M, Gao X, Su JZ, Nie S (2001) Quantum-dot-tagged microbeads for multiplexed optical coding of biomolecules. Nat Biotechnol 19(7):631–635. doi: 10.1038/9022890228 PubMedCrossRefGoogle Scholar
  40. 40.
    Law WC, Yong KT, Roy I, Ding H, Hu R, Zhao W, Prasad PN (2009) Aqueous-phase synthesis of highly luminescent CdTe/ZnTe core/shell quantum dots optimized for targeted bioimaging. Small 5(11):1302–1310. doi: 10.1002/smll.200801555 PubMedCrossRefGoogle Scholar
  41. 41.
    Algarra M, Campos BB, Miranda MS, da Silva JC (2011) CdSe quantum dots capped PAMAM dendrimer nanocomposites for sensing nitroaromatic compounds. Talanta 83(5):1335–1340. doi: 10.1016/j.talanta.2010.10.056 PubMedCrossRefGoogle Scholar
  42. 42.
    Charbonniere LJ, Hildebrandt N, Ziessel RF, Lohmannsroben HG (2006) Lanthanides to quantum dots resonance energy transfer in time-resolved fluoro-immunoassays and luminescence microscopy. J Am Chem Soc 128(39):12800–12809. doi: 10.1021/ja062693a PubMedCrossRefGoogle Scholar
  43. 43.
    Azzazy HM, Mansour MM, Kazmierczak SC (2006) Nanodiagnostics: a new frontier for clinical laboratory medicine. Clin Chem 52(7):1238–1246. doi: 10.1373/clinchem.2006.066654 PubMedCrossRefGoogle Scholar
  44. 44.
    Wang HQ, Huang ZL, Liu TC, Wang JH, Cao YC, Hua XF, Li XQ, Zhao YD (2007) A feasible and quantitative encoding method for microbeads with multicolor quantum dots. J Fluoresc 17(2):133–138. doi: 10.1007/s10895-007-0157-5 PubMedCrossRefGoogle Scholar
  45. 45.
    Morgner F, Stufler S, Geissler D, Medintz IL, Algar WR, Susumu K, Stewart MH, Blanco-Canosa JB, Dawson PE, Hildebrandt N (2011) Terbium to quantum Dot FRET bioconjugates for clinical diagnostics: influence of human plasma on optical and assembly properties. Sensors (Basel) 11(10):9667–9684. doi: 10.3390/s111009667 CrossRefGoogle Scholar
  46. 46.
    Charbonnière LJ, Hildebrandt N (2008) Lanthanide complexes and quantum dots: a bright wedding for resonance energy transfer. European Jounal of Inorganic Chmistry 2008(21):3241–3251. doi: 10.1002/ejic.200800332 CrossRefGoogle Scholar
  47. 47.
    Yu WW, Qu L, Guo W, Peng X (2003) Experimental determination of the extinction coefficient of CdTe, CdSe, and CdS nanocrystals. Chem Mater 15(14):2854–2860. doi: 10.1021/cm034081k CrossRefGoogle Scholar
  48. 48.
    Liu TC, Huang ZL, Wang HQ, Wang JH, Li XQ, Zhao YD, Luo QM (2006) Temperature-dependent photoluminescence of water-soluble quantum dots for a bioprobe. Anal Chim Acta 559(1):120–123. doi: 10.1016/j.aca.2005.11.053 CrossRefGoogle Scholar
  49. 49.
    Freeman R, Liu X, Willner I (2011) Amplified multiplexed analysis of DNA by the exonuclease III-catalyzed regeneration of the target DNA in the presence of functionalized semiconductor quantum dots. Nano Lett 11(10):4456–4461. doi: 10.1021/nl202761g PubMedCrossRefGoogle Scholar
  50. 50.
    Dong ZN, Wu YS, Wang Z, He A, Li M, Chen M, Du H, Ma Q, Liu T (2012) Effect of temperature on the photoproperties of luminescent terbium sensors for homogeneous bioassays. Luminescence. doi: 10.1002/bio.2355
  51. 51.
    Kubinm RF, Fletcher AN (1982) Fluorescence quantum yields of some rhodamine dyes. J Luminescence 27:455–462CrossRefGoogle Scholar
  52. 52.
    Hemmilä IA, Mikola HJ (1990) New complexing agents for labeling of proteins with metals. Acta Radiol Suppl 374:53–55PubMedGoogle Scholar
  53. 53.
    Hemmilä I, Mukkala V-M, Takalo H (1997) Development of luminescent lanthanide chelate labels for diagnostic assays. J Alloys Comp 249(1–2):158–162. doi: 10.1016/S0925-8388(96)02834-4 CrossRefGoogle Scholar
  54. 54.
    Hemmilä I, Dakubu S, Mukkala V-M, Siitari H, Lövgren T (1984) Europium as a label in time-resolved immunofluorometric assays. Anal Biochem 137(2):335–343PubMedCrossRefGoogle Scholar
  55. 55.
    Wang J, Jiang P, Han Z, Qiu L, Wang C, Zheng B, Xia J (2012) Fast self-assembly kinetics of quantum dots and a dendrimeric peptide ligand. Langmuir 28(21):7962–7966. doi: 10.1021/la301227r PubMedCrossRefGoogle Scholar
  56. 56.
    Hildebrandt N, Geissler D (2012) Semiconductor quantum dots as FRET acceptors for multiplexed diagnostics and molecular ruler application. Adv Exp Med Biol 733:75–86. doi: 10.1007/978-94-007-2555-3_8 PubMedCrossRefGoogle Scholar
  57. 57.
    Perez-Donoso JM, Monras JP, Bravo D, Aguirre A, Quest AF, Osorio-Roman IO, Aroca RF, Chasteen TG, Vasquez CC (2012) Biomimetic, mild chemical synthesis of CdTe-GSH quantum dots with improved biocompatibility. PLoS One 7(1):e30741. doi: 10.1371/journal.pone.0030741 PubMedCrossRefGoogle Scholar
  58. 58.
    Schulman SG (1977) Fluorescence and phosphorescence spectroscopy: physicochemical principles and practice. Pergamon, OxfordGoogle Scholar
  59. 59.
    Valeur B (2002) Molecular fluorescence: principles and applications. Wiley-VCH, Weinheim; ChichesterGoogle Scholar
  60. 60.
    Lakowicz JR (1999) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic/Plenum, New York; LondonCrossRefGoogle Scholar
  61. 61.
    Morgner F, Geissler D, Stufler S, Butlin NG, Lohmannsroben HG, Hildebrandt N (2010) A quantum-dot-based molecular ruler for multiplexed optical analysis. Angew Chem Int Ed Engl 49(41):7570–7574. doi: 10.1002/anie.201002943 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Zhen-Hua Chen
    • 1
  • Ying-Song Wu
    • 1
  • Mei-Jun Chen
    • 1
  • Jing-Yuan Hou
    • 1
  • Zhi-Qi Ren
    • 1
  • Da Sun
    • 1
  • Tian-Cai Liu
    • 1
  1. 1.School of BiotechnologySouthern Medical UniversityGuangzhouPeople’s Republic of China

Personalised recommendations