Synthesis of quantum dots and their complexes for multiplexed cancer marker imaging and ratiometric oxygen sensing

Abstract

Semiconductor nanocrystal, namely quantum dots (QDs) have many unique properties such as bright photoluminescence, capability of multiplexed imaging and resistance against photobleaching. These properties of QD make it proper constant agent in biomedical application fields. Emission wavelength of QDs can be easily tunable from visible to infrared wavelength range by changing the size or compositions of QDs. To use QDs in bio-imaging fields, it is important to synthesize high quality core/shell QDs. In this research, for QDs emitting red, yellow, and green lights, the size of QDs was controlled by reaction conditions, such as injection temperature and precursor concentration. And QDs which emit blue light were synthesized by changing composition of QDs. Core/Shell QDs synthesized in organic solvent have high quality like good crystallinity and high photoluminescence quantum yield (PLQY), because their surfaces are passivated with hydrophobic ligands. By stacking thick shell layers onto QDs, QDs could obtain high optical properties. However, since the surface of QD is surrounded with hydrophobic ligands, QD must be transferred to aqueous phase for using in biological environment. In this research, two methods (surface ligand exchange and encapsulation of QDs with amphiphilic polymer) were used to transfer QDs to aqueous phase. First, the surface of QD was modified by zwitterionic ions. After surface ligand exchange, QDs meet good colloidal stability, minimal hydrodynamic size, and capability of conjugation with other functional groups. To prepare multiplexed QD probes for diagnosis of colon cancer, QDs which emit red, yellow, and green light were conjugated to colon cancer markers of matrix metalloproteinase (MMP) 9, MMP 14, and carcinoembryonic antigen (CEA) antibodies (Abs) with zwitterionic surface coating for minimal non-specific bindings, respectively. CEA, MMP 9 and MMP 14 actively implicate cancer progression and metastasis. The multicolor detection by using antibody(Ab)-QD conjugates (Ab-QD probes) is expected to have potential for rapid and accurate colon cancer diagnosis. As the second method, to transfer QDs to aqueous phase, QDs were encapsulated with amphiphilic polymer (PEI-HDI) which was formed with polyethyleneimine (PEI) and hexadecyl isocyanate (HDI). Encapsulated QDs meet colloidal stability in aqueous phase. And the encapsulated QDs have low toxicity and high PLQY. In this work, oxygen probe (QD-Ru-amPEI) was synthesized by encapsulating QDs and oxygen sensitive phosphorescence dye (Tris(4,7-diphenyl-1,10-phenanthroline)-ruthenium hexafluorophosphate, Ru dye) together with PEI-HDI to measure amount of oxygen in tumor. Oxygen is a critical factor to damage DNA of cancer cells by fixation radical on DNA in radiation therapy. But, since most of cancer cells generally divide rapidly and are unregulated, they can form solid tumors which are big enough to cause deficiency of oxygen in central cells of solid tumors. So, developing the sensitive oxygen probe which can measure amount of oxygen is very important in radiation therapy. By encapsulating QDs and Ru dye with PEI-HDI, the distance between QDs and Ru dye was close enough to induce Förster-type resonance energy transfer (FRET) efficiently. When the Ru dye is confronted with oxygen, its phosphorescence intensity and lifetime decrease. Since QDs have broad absorption profile with two-photon absorption (TPA) cross-sections, QDs were utilized as TPA antenna. When QDs were excited, QD’s fluorescence and Ru dye’s phosphorescence were emitted. QD’s fluorescence was used as a standard because of its insensitive fluorescence to oxygen. Through the photoluminescence ratio of QD to Ru, the relative amount of oxygen could be calculated in spheroid cells which were formed by cancer cells to mimic real environment of cancer cells in body.