Advanced multi-photon imaging and diagnostics with quantum dot-based hybrid nanostructures.
Advanced multi-photon imaging and diagnostics with quantum dot-based hybrid nanostructures
Igor Nabiev a,b,*
a Laboratory of Nano-Bioengineering, National Research Nuclear University MEPhI (Moscow Engineering Physics Institute), 115409 Moscow, Russian Federation
b Laboratoire de Recherche en Nanosciences, EA4682-LRN, Université de Reims Champagne-Ardenne, 51100 Reims, France
Cancer remains one of the leading causes of death in the world. The use of a panel of proven biomarkers (instead of single biomarkers) can significantly improve the sensitivity and efficiency of diagnosis. Advanced multiplexed imaging with quantum dots (QDs) may become an indispensable tool for early cancer diagnostics1.
An ideal single- or multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest single-photon (1P) or two-photon (2P) absorption cross section (ACS) and the smallest highly specific recognition molecules conjugated with a nanoparticle in an oriented manner2,3. However, the conditions used for conjugation of typical recognition molecules (conventional antibodies, Abs) with nanoparticles often provoke their unfolding and yield nanoprobes with irregular orientation of Abs on the nanoparticle surface. Conjugation of smaller Ab fragments, such as single-domain antibodies (sdAbs), with QDs in an oriented manner can be considered as an attractive approach to generation of ultrasmall diagnostic nanoprobes2.
CdSe/ZnS QDs conjugated to 13-kDa sdAbs derived from camelid IgG or streptavidin have been used as efficient 1P- or 2P-excitation probes for imaging of carcinoembryonic antigen (CEA), HER2, and other biomarkers2-4. The 2P-ACS for some conjugates is higher than 49,000 GM (Goeppert-Mayer units), which is close to the theoretical value calculated for CdSe QDs (50,000 GM)3 and considerably exceeds that of organic dyes.
High-quality imaging in medical practice requires clear discrimination between the fluorescence of immunostaining probes and the autofluorescence of biological liquids and tissues. 2P-microscopy with excitation in the near-IR spectral region has now become the main fluorescence imaging technique for thick biological specimens, because the excitation band is far from the spectral region of tissue autofluorescence excitation and enables deep imaging of biological tissues.
Figure 1. Two-photon (2P) excitation of the immunostaining complexes for MC38 cells overexpressing carcinoembryonic antigen (CEA).
The sample is labeled with oriented conjugates of quantum dots and single-domain antibodies (sdAbs) and, additionally, with a primary antibody labeled with the AlexaFluor dye forming an efficient donor/acceptor pair with the QD.
A further step in this direction has been made in our study (Figure 1). Here, efficient FRET under 2P excitation was demonstrated for the double immunostaining complexes formed on the surface of MC38 cancer cells overexpressing CEA with the use of sdAb-QD conjugates (donor) and a combination of the monoclonal Ab against CEA and the secondary antibody labeled with the AlexaFluor dye (acceptor).
The proposed approach permits obtaining an exceptional contrast of 2P imaging of cancer biomarkers without any contribution of cell and tissue autofluorescence in the recorded images thus paving a way to advanced diagnostics with the use of highly oriented conjugates of sdAbs with the colloidal nanomaterials.
- K. Brazhnik, Z. Sokolova, M. Baryshnikova, R. Bilan, A. Efimov, I. Nabiev, A. Sukhanova, Quantum dot-based lab-on-a-bead system for multiplexed detection of free and total prostate-specific antigens in clinical human serum samples. Nanomedicine 11, 5, 1065–1075 (2015)
- A. Sukhanova, K. Even-Desrumeaux, A. Kisserli, T. Tabary, B. Reveil, J.M. Millot, P. Chames, D. Baty, M. Artemyev, S. Poly, V. Oleinikov, M. Pluot, J.H.M. Cohen, I. Nabiev, Oriented conjugates single-domain antibodies and quantum dots: Toward new generation of ultra-small diagnostic nanoprobes. Nanomedicine 8, 516–525 (2012)
- H. Hafian, A. Sukhanova, M. Turini, P. Chames, D. Baty, M. Pluot, J.H.M. Cohen, I. Nabiev, I., J.M. Millot, Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots. Nanomedicine 10, 8, 1701–1709 (2014)
- T.Y. Rakovich, O.K. Mahfoud, B.M. Mohamed, A. Prina-Mello, K. Crosbie-Staunton, T. Van Den Broeck, L. De Kimpe, A. Sukhanova, et al., Highly sensitive single domain antibody-quantum dot conjugates for detection of low expression levels of HER2 biomarker in lung and breast cancer cells. ACS NANO 8, 6, 5682–5695 (2014).