Analytical and Bioanalytical Chemistry

, Volume 400, Issue 2, pp 369–379 | Cite as

Conjugation reactions in the preparations of quantum dot-based immunoluminescent probes for analysis of proteins by capillary electrophoresis

  • M. Lišková
  • I. Voráčová
  • K. KlepárníkEmail author
  • V. Hezinová
  • J. Přikryl
  • F. Foret
Original Paper


A number of biologically important molecules, such as DNA, proteins, and antibodies, are routinely conjugated with fluorescent tags for high-sensitivity analyses. Here, the application of quantum dots in the place of bright and size-tunable luminophores is studied. Several selected bioconjugation reactions via zero-length cross-linkers, long-chain linkers, and oriented methods for linking of quantum dots with proteins were tested. Anti-ovalbumin, anti-proliferating cell nuclear antigen, anti-hemagglutinin, and anti-CD3 membrane protein as model antibodies and annexin V were used as high-specificity selectors. The reaction yield and efficiency of the prepared immunoluminescent probes were tested by capillary zone electrophoresis with laser-induced fluorescence detection.


Scheme of antibody-quantum dot conjugate


CdTe quantum dots Luminescent probe Capillary zone electrophoresis Bioconjugation reactions 



The authors wish to thank Dr. Borivoj Vojtesek from Masaryk Memorial Cancer Institute for the kind gift of a purified anti-hemagglutinin. This work was supported by the following grants: The Grant Agency of the Czech Republic number 203/08/1680 and 301/11/2055, Ministry of Education, Youth and Sports number LC06023, bilateral project CZ-ES 2009-10/6 (2008CZ0017) and institute research plan AV0Z40310501.


  1. 1.
    Heegaard NHH, Kennedy RT (2002) Antigen–antibody interactions in capillary electrophoresis. J Chromatogr B Anal Technol Biomed Life Sci 768(1):93–103CrossRefGoogle Scholar
  2. 2.
    Schultz NM, Kennedy RT (1993) Rapid immunoassays using capillary electrophoresis with fluorescence detection. Anal Chem 65(21):3161–3165CrossRefGoogle Scholar
  3. 3.
    Schmalzing D, Buonocore S, Piggee C (2000) Capillary electrophoresis-based immunoassays. Electrophoresis 21(18):3919–3930CrossRefGoogle Scholar
  4. 4.
    Schmalzing D, Nashabeh W (1997) Capillary electrophoresis based immunoassays: a critical review. Electrophoresis 18(12–13):2184–2193CrossRefGoogle Scholar
  5. 5.
    Yang WC, Schmerr MJ, Jackman R, Bodemer W, Yeung ES (2005) Capillary electrophoresis-based noncompetitive immunoassay for the prion protein using fluorescein-labeled protein A as a fluorescent probe. Anal Chem 77(14):4489–4494CrossRefGoogle Scholar
  6. 6.
    Shimura K, Karger BL (1994) Affinity probe capillary electrophoresis—analysis of recombinant human growth-hormone with a fluorescent-labeled antibody fragment. Anal Chem 66(1):9–15CrossRefGoogle Scholar
  7. 7.
    Ma J, Chen JY, Guo J, Wang CC, Yang WL, Xu L, Wang PN (2006) Photostability of thiol-capped CdTe quantum dots in living cells: the effect of photo-oxidation. Nanotechnology 17(9):2083–2089CrossRefGoogle Scholar
  8. 8.
    Eychmuller A, Rogach AL (2000) Chemistry and photophysics of thiol-stabilized II–VI semiconductor nanocrystals. Pure Appl Chem 72(1–2):179–188CrossRefGoogle Scholar
  9. 9.
    Lakowicz J (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer Science, New YorkCrossRefGoogle Scholar
  10. 10.
    Masumoto Y, Sonobe K (1997) Size-dependent energy levels of CdTe quantum dots. Phys Rev B 56(15):9734–9737CrossRefGoogle Scholar
  11. 11.
    Lee J, Choi Y, Kim K, Hong S, Park HY, Lee T, Cheon GJ, Song R (2010) Characterization and cancer cell specific binding properties of anti-EGFR antibody conjugated quantum dots. Bioconjug Chem 21(5):940–946CrossRefGoogle Scholar
  12. 12.
    van Embden J, Jasieniak J, GĂłmez DE, Mulvaney P, Giersig M (2007) Review of the synthetic chemistry involved in the production of core/shell semiconductor nanocrystals. Aust J Chem 60(7):457–471CrossRefGoogle Scholar
  13. 13.
    Hua XF, Liu TC, Cao YC, Liu B, Wang HQ, Wang JH, Huang ZL, Zhao YD (2006) Characterization of the coupling of quantum dots and immunoglobulin antibodies. Anal Bioanal Chem 386(6):1665–1671CrossRefGoogle Scholar
  14. 14.
    Schroedter A, Weller H, Eritja R, Ford WE, Wessels JM (2002) Biofunctionalization of silica-coated CdTe and gold nanocrystals. Nano Lett 2(12):1363–1367CrossRefGoogle Scholar
  15. 15.
    Wolcott A, Gerion D, Visconte M, Sun J, Schwartzberg A, Chen SW, Zhang JZ (2006) Silica-coated CdTe quantum dots functionalized with thiols for bioconjugation to IgG proteins. J Phys Chem B 110(11):5779–5789CrossRefGoogle Scholar
  16. 16.
    Yang P, Zhang AY, Sun HS, Liu FT, Jiang QH, Cheng X (2010) Highly luminescent quantum dots functionalized and their conjugation with IgG. J Colloid Interface Sci 345(2):222–227CrossRefGoogle Scholar
  17. 17.
    Dong W, Ge X, Wang M, Xu SK (2010) Labeling of BSA and imaging of mouse T-lymphocyte as well as mouse spleen tissue by l-glutathione capped CdTe quantum dots. Luminescence 25(1):55–60Google Scholar
  18. 18.
    Wang JH, Wang HQ, Zhang HL, Li XQ, Hua XF, Cao YC, Huang ZL, Zhao YD (2007) Purification of denatured bovine serum albumin coated CdTe quantum dots for sensitive detection of silver(I) ions. Anal Bioanal Chem 388(4):969–974CrossRefGoogle Scholar
  19. 19.
    Klostranec JM, Chan WCW (2006) Quantum dots in biological and biomedical research: recent progress and present challenges. Adv Mater 18(15):1953–1964CrossRefGoogle Scholar
  20. 20.
    Burda C, Chen XB, Narayanan R, El-Sayed MA (2005) Chemistry and properties of nanocrystals of different shapes. Chem Rev 105(4):1025–1102CrossRefGoogle Scholar
  21. 21.
    Medintz IL, Uyeda HT, Goldman ER, Mattoussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4(6):435–446CrossRefGoogle Scholar
  22. 22.
    Pyell U (2010) Characterization of nanoparticles by capillary electromigration separation techniques. Electrophoresis 31(5):814–831CrossRefGoogle Scholar
  23. 23.
    Pereira M, Lai E (2008) Capillary electrophoresis for the characterization of quantum dots after non-selective or selective bioconjugation with antibodies for immunoassay. J Nanobiotechnol 6(1):10CrossRefGoogle Scholar
  24. 24.
    Xing Y, Chaudry Q, Shen C, Kong KY, Zhau HE, Chung LW, Petros JA, O'Regan RM, Yezhelyev MV, Simons JW, Wang MD, Nie S (2007) Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat Protoc 2(5):1152–1165CrossRefGoogle Scholar
  25. 25.
    Ma Q, Wang XY, Li YB, Su XG, Jin QH (2007) The use of COP quantum dot fluorescent microspheres in fluoro-immunoassays and a microfluidic chip system. Luminescence 22(5):438–445CrossRefGoogle Scholar
  26. 26.
    Wang XY, Ma Q, Li YB, Li B, Su XG, Jin QH (2005) Studies on fluorescence resonance energy transfer between dyes and water-soluble quantum dots. Can J Anal Sci Spectros 50(3):141–146Google Scholar
  27. 27.
    Lei Y, Jiang CY, Liu SJ, Miao YM, Zou BS (2006) A clean route for preparation of CdTe nanocrystals and their conjugation with bacterium. J Nanosci Nanotechnol 6(12):3784–3788CrossRefGoogle Scholar
  28. 28.
    Xue FL, Chen JY, Guo J, Wang CC, Yang WL, Wang PN, Lu DR (2007) Enhancement of intracellular delivery of CdTe quantum dots (QDs) to living cells by Tat conjugation. J Fluoresc 17(2):149–154CrossRefGoogle Scholar
  29. 29.
    Yang H, Guo Q, He R, Li D, Zhang XQ, Bao CC, Hu HY, Cui DX (2009) A quick and parallel analytical method based on quantum dots labeling for ToRCH-related antibodies. Nanoscale Res Lett 4(12):1469–1474CrossRefGoogle Scholar
  30. 30.
    Hermanson G (1996) Bioconjugate techniques. Elsevier, San DiegoGoogle Scholar
  31. 31.
    Grabarek Z, Gergely J (1990) Zero-length crosslinking procedure with the use of active esters. Anal Biochem 185(1):131–135CrossRefGoogle Scholar
  32. 32.
    Qian J, Zhang CY, Cao XD, Liu SQ (2010) Versatile immunosensor using a quantum dot coated silica nanosphere as a label for signal amplification. Anal Chem 82(15):6422–6429CrossRefGoogle Scholar
  33. 33.
    Tang B, Cao LH, Xu KH, Zhuo LH, Ge JH, Li QF, Yu LJ (2008) A new nanobiosensor for glucose with high sensitivity and selectivity in serum based on fluorescence resonance energy transfer (FRET) between CdTe quantum dots and an nanoparticles. Chem Eur J 14(12):3637–3644CrossRefGoogle Scholar
  34. 34.
    Zeng QH, Zhang YL, Song K, Kong XG, Aalders MCG, Zhang H (2009) Enhancement of sensitivity and specificity of the fluoroimmunoassay of hepatitis B virus surface antigen through “flexible” coupling between quantum dots and antibody. Talanta 80(1):307–312CrossRefGoogle Scholar
  35. 35.
    Sun P, Zhang HY, Liu C, Fang J, Wang M, Chen J, Zhang JP, Mao CB, Xu SK (2010) Preparation and characterization of Fe3O4/CdTe magnetic/fluorescent nanocomposites and their applications in immuno-labeling and fluorescent imaging of cancer cells. Langmuir 26(2):1278–1284CrossRefGoogle Scholar
  36. 36.
    Balamurugan S, Obubuafo A, Soper SA, Spivak DA (2008) Surface immobilization methods for aptamer diagnostic applications. Anal Bioanal Chem 390(4):1009–1021CrossRefGoogle Scholar
  37. 37.
    Pathak S, Choi S-K, Arnheim N, Thompson ME (2001) Hydroxylated quantum dots as luminescent probes for in situ hybridization. J Am Chem Soc 123(17):4103–4104CrossRefGoogle Scholar
  38. 38.
    Riegler J, Ehlert O, Nann T (2006) A facile method for coding and labeling assays on polystyrene beads with differently colored luminescent nanocrystals. Anal Bioanal Chem 384(3):645–650CrossRefGoogle Scholar
  39. 39.
    Wang JH, Li YQ, Zhang HL, Wang HQ, Lin S, Chen J, Zhao YD, Luo QM (2010) Bioconjugation of concanavalin and CdTe quantum dots and the detection of glucose. Colloids Surf, A Physicochem Eng Asp 364(1–3):82–86CrossRefGoogle Scholar
  40. 40.
    O'Shannessy DJ (1990) Hydrazido-derivatized supports in affinity chromatography. J Chromatogr A 510:13–21CrossRefGoogle Scholar
  41. 41.
    Bilkova Z, Mazurova J, Churacek J, Horak D, Turkova J (1999) Oriented immobilization of chymotrypsin by use of suitable antibodies coupled to a nonporous solid support. J Chromatogr A 852(1):141–149CrossRefGoogle Scholar
  42. 42.
    Walther C, Meyer K, Rennert R, Neundorf I (2008) Quantum dot–carrier peptide conjugates suitable for imaging and delivery applications. Bioconjug Chem 19(12):2346–2356CrossRefGoogle Scholar
  43. 43.
    Rogach AL, Katsikas L, Kornowski A, Su DS, Eychmuller A, Weller H (1996) Synthesis and characterization of thiol-stabilized CdTe nanocrystals. Ber Bunsen-Ges Phys Chem Chem Phys 100(11):1772–1778Google Scholar
  44. 44.
    Klayman DL, Griffin TS (1973) Reaction of selenium with sodium-borohydride in protic solvents—facile method for introduction of selenium into organic-molecules. J Am Chem Soc 95(1):197–200CrossRefGoogle Scholar
  45. 45.
    Zhang J, Badugu R, Lakowicz J (2008) Fluorescence quenching of CdTe nanocrystals by bound gold nanoparticles in aqueous solution. Plasmonics 3(1):3–11CrossRefGoogle Scholar
  46. 46.
    AB Genovis. Accessed 4 January 2011
  47. 47.
    Kimura K, Takashima S, Ohshima H (2002) Molecular approach to the surface potential estimate of thiolate-modified gold nanoparticles. J Phys Chem B 106(29):7260–7266CrossRefGoogle Scholar
  48. 48.
    Eaton DF (1988) Reference materials for fluorescence measurement. Pure Appl Chem 60(7):1107–1114CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • M. Lišková
    • 1
  • I. Voráčová
    • 1
  • K. Klepárník
    • 1
    Email author
  • V. Hezinová
    • 1
  • J. Přikryl
    • 1
  • F. Foret
    • 1
  1. 1.Institute of Analytical ChemistryAcademy of Sciences of the Czech RepublicBrnoCzech Republic

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