Hostname: page-component-6b989bf9dc-g5k2d Total loading time: 0 Render date: 2024-04-15T02:43:21.583Z Has data issue: false hasContentIssue false

Bioconjugated and Cross-Linked Bionanostructures for Bifunctional Immunohistochemical Labeling

Published online by Cambridge University Press:  13 March 2012

Rıdvan Say*
Department of Chemistry, Anadolu University, Eskişehir, Turkey
Gözde Aydoğan Kılıç
Department of Biology, Anadolu University, Eskişehir, Turkey
Ayça Atılır Özcan
Department of Chemistry, Anadolu University, Eskişehir, Turkey
Deniz Hür
Department of Chemistry, Anadolu University, Eskişehir, Turkey Plant, Drug and Scientific Research Center, Anadolu University, Eskişehir, Turkey
Filiz Yılmaz
Department of Chemistry, Anadolu University, Eskişehir, Turkey
Adil Denizli
Department of Chemistry, Hacettepe University, Ankara, Turkey
Arzu Ersöz
Department of Chemistry, Anadolu University, Eskişehir, Turkey
Corresponding author. E-mail:
Get access


The present study describes the development and use of a new bioconjugate combining targeted quantum dot labeling with an immunoperoxidase method and explores whether these bioconjugates could specifically and effectively label Cu/Zn superoxide dismutase (SOD1). The new bioconjugate is designed for the examination of samples both under fluorescent and bright-field microscopy at the same time. For this purpose chlorobis(2-2′-bipyridyl) methacryloyl tyrosine-ruthenium(II) and bis (2-2′-bipyridyl) methacryloyltyrosine-methacryloyltryptophan-ruthenium (II) photosensitive monomers and photosensitive poly(Bis (2-2′-bipyridyl)) methacryloyltyrosine-methacryloyltryptophan-ruthenium(II) were synthesized and characterized. The anti-SOD1 antibody and horseradish peroxidase (HRD) conjugated quantum dots were prepared by using this polymer. The anti-SOD1 antibody and HRD conjugated quantum dots were used in labeling and imaging of SOD1 in rat liver sections. Quantum dot particles were observed as a bright fluorescence in their specific binding locations inside the hepatocytes. The HRD-diaminobenzidine reaction product was observed as brown-colored particles at the same locations under bright-field microscopy. Structural details of the tissue sections could be examined at the same time. The conjugation protocol is simple; the bioconjugate is applicable for efficient cell labeling and can be adapted for imaging of other targets in different tissues. Also, the prepared nanobioconjugates have mechanic stability and can be used for a long period.

Biological and Biomedical Applications
Copyright © Microscopy Society of America 2012

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)


Chang, L.Y., Slot, W.J., Geuze, H.J. & Crapo, J.D. (1988). Molecular immunocytochemistry of the CuZn superoxide dismutase in rat hepatocytes. J Cell Biol 107(6), 21692179.CrossRefGoogle ScholarPubMed
Chou, L.Y.T., Fischer, H.C., Perrault, S.D. & Chan, W.C.W. (2009). Visualizing quantum dots in biological samples using silver staining. Anal Chem 81, 45604565.CrossRefGoogle ScholarPubMed
Deerinck, T.J. (2008). The application of fluorescent quantum dots to confocal, multiphoton, and electron microscopic imaging. Toxicol Pathol 36, 112116.CrossRefGoogle ScholarPubMed
Defrus, A.M., Chen, A.A., Min, D.H., Ruoslahti, E. & Bhaita, S.N. (2007). Targeted quantum dot conjugates for siRNA Delivery. Bioconjugate Chem 18, 13911396.Google Scholar
Diltemiz, S.E., Say, R., Büyüktiryaki, S., Hür, D., Denizli, A. & Ersöz, A. (2008). Quantum dot nanocrystals having guanosineimprinted nanoshell for DNA recognition. Talanta 75, 890896.CrossRefGoogle ScholarPubMed
Evans, I.P., Spencer, A. & Wilkinson, G. (1973). Dichlorotetrakis (dimethyl sulphoxide) ruthenium (II) and its use as a source material for some new ruthenium (II) complexes. J Chem Soc Dalton Trans 204209.CrossRefGoogle Scholar
Frangioni, J.V. (2003). In vivo near-infrared fluorescence imaging. Curr Opin Chem Biol 7, 626634.CrossRefGoogle ScholarPubMed
Gilbert, E.S., Khlebnikov, A., Cowan, S.E. & Keasling, J.D. (2001). Analysis of biofilm structure and gene expression using fluorescence dual labeling. Biotechnol Prog 17, 11801182.CrossRefGoogle ScholarPubMed
Grube, D. (1980). Immunoperoxidase methods: Increased efficiency using fluorescence microscopy for 3,3-diaminobenzidine (DAB) stained semithin sections. Histochemistry 70, 1922.CrossRefGoogle ScholarPubMed
Hür, D., Ekti, S.F. & Say, R. (2007). N-Acylbenzotriazole mediated synthesis of some methacrylamido amino acids. Lett Org Chem 4(8), 585587.CrossRefGoogle Scholar
Kawamata, H. & Manfredi, G. (2008). Different regulation of wild-type and mutant Cu,Zn superoxide dismutase localization in mammalian mitochondria. Hum Mol Gen 17(21), 33033317.CrossRefGoogle ScholarPubMed
Kruttwig, K., Bruggemann, C., Kaijzel, E., Vorhagen, S., Hilger, T., Löwik, C. & Hoehn, M. (2010). Development of a three-dimensional in vitro model for longitudinal observation of cell behavior: Monitoring by magnetic resonance imaging and optical imaging. Mol Imaging Biol 12, 367376.CrossRefGoogle ScholarPubMed
Kulkarni, S.K., Ethiraj, A.S., Kharrazi, S., Deobagkar, D.N. & Deobagkar, D.D. (2005). Synthesis and spectral properties of DNA capped CdS nanoparticles in aqueous and non-aqueous media. Biosens Bioelect 21, 95102.CrossRefGoogle ScholarPubMed
Okado-Matsumoto, A. & Fridovich, I. (2001). Subcellular distribution of superoxide dismutases in rat liver: Cu,Zn-SOD in mitochondria. J Biol Chem 276(42), 3838838393.CrossRefGoogle ScholarPubMed
Ornberg, R.L., Wu, X. & Bruchez, M.P. (2004). Qdot conjugates: A novel fluorescence detection technology for biological imaging. Microsc Microanal 10, 12721273.CrossRefGoogle Scholar
Parak, W.J., Pellegrino, T. & Plank, C. (2005). Labelling of cells with quantum dots. Nanotechnology 16, R9R25.CrossRefGoogle ScholarPubMed
Robertson, D., Savage, K., Reis-Filho, J.S. & Isacke, C.M. (2008). Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. BMC Cell Biol 9, 1322.CrossRefGoogle ScholarPubMed
Say, R. (2011). Photosensitive amino acid-monomer linkage and bioconjugation applications in life sciences and biotechnology. World Intellectual Property Organization–PatentScope, (Pub. No.: WO/2011/070402; Int. Appl. No.: PCT/IB2009.055707).Google Scholar
Say, R., Aydoğan Kiliç, G., Atilir Özcan, A., Hür, D., Yilmaz, F., Kutlu, M., Yazar, S., Denizli, A., Emir Diltemiz, S. & Ersöz, A. (2011). Investigation of photosensitively bioconjugated targeted quantum dots for the labeling of Cu/Zn superoxide dismutase in fixed cells and tissue sections. Histochem Cell Biol 135(5), 523530.CrossRefGoogle ScholarPubMed
Stelter, L., Pinkernelle, J.G., Michel, R., Schwartlander, R., Raschzok, N., Morgul, M.H., Koch, M., Denecke, T., Ruf, J., Baumler, H., Jordan, A., Hamm, B., Sauer, I.M. & Teichgraber, U. (2010). Modification of aminosilanized superparamagnetic nanoparticles: Feasibility of multimodal detection using 3T MRI, Small animal PET, and fluorescence imaging. Mol Imaging Biol 12, 2534.CrossRefGoogle ScholarPubMed
Sweeney, E., Ward, T.H., Gray, N., Womack, C., Jayson, G., Hughes, A., Dive, C. & Byers, R. (2008). Quantitative multiplexed quantum dot immunohistochemistry. Biochem Biophys Res Commun 374, 181186.CrossRefGoogle ScholarPubMed
Taylor, C.R. (1978). Immunoperoxidase techniques practical and theorical aspects. Arch Pathol Lab Med 102(3), 113121.Google Scholar
Wang, H.Z., Wang, H.Y., Lıang, R.Q. & Ruan, K.C. (2004). Detection of tumor marker CA125 in ovarian carcinoma using quantum dots. Acta Biochim Biophys Sin 36(10), 681686.CrossRefGoogle ScholarPubMed
Xing, Y., Chaudry, Q., Shen, C., Kong, K.Y., Zhau, H.E., Chung, L.W., Petros, J.A., O'Regan, R.M., Yezhelyev, M.V., Sımons, J.W., Wang, M.D. & Nıe, S. (2007). Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry. Nat Prot 2(5), 11521165.CrossRefGoogle ScholarPubMed
Xu, Y., Wang, Q., He, P., Dong, Q., Lıu, F., Lıu, Y., Lın, L., Yan, H. & Zhao, X. (2008). Cell nucleus penetration by quantum dots induced by nuclear staining organic fluorophore and UV-irradiation. Adv Mater 20, 34683473.CrossRefGoogle Scholar
Yoo, J., Kambara, T., Gonda, K. & Higuchi, H. (2008). Intracellular imaging of targeted proteins labeled with quantum dots. Exp Cell Res 314, 35633569.CrossRefGoogle ScholarPubMed
Yu, X., Chen, L., Deng, Y., Li, K., Wang, Q., Li, Y., Xiao, S., Zhou, L., Luo, X., Liu, J. & Pang, D. (2007). Fluorescence analysis with quantum dot probes for hepatoma under one- and two-photon excitation. J Fluoresc 17, 243247.CrossRefGoogle ScholarPubMed