Purification Of CdSe@ZnS Quantum Dot-Labeled Antibody Bioprobes And Applications In Tumor Marker Detection

Quantum dots (QDs) are inorganic semi-conductor nano-crystals with excellent optical properties including wide excitation spectrum, narrow emission spectrum, high quantum yield, great photostability, and so on. So it has been widely applied in biomedicine field such as sensing, labeling, imaging, and tracking. Various strategies of chemosynthesis and biosynthesis of QDs have been developed in last two decades. High quality CdSe@ZnS QDs are prepared in the organic phase by chemosynthesis till now. However, hydrophobic QDs are inappropriate for bioimaging. Therefore, transferring QDs from non-polar solvents to aqueous phase and further functionalization is the prerequisite for the development of QD-based bioapplications. To address this issue, ligand exchange and hydrophobic encapsulation have reported for QDs surface engineering to render QDs water dispersity. The encapsulation strategy, namely coating of QDs with amphiphilic compounds, such as amphiphilic polymers, is more favorable because the fluorescence intensity of hydrophilic QDs could be completely retained. However, aggregated QDs would destroy the balance of colloidal stability of QD-based bioprobes, and the excess functional biomacromolecule and coupling reagent for modification should be removed off. Colloidal impurities such as suspended agglomerates of QDs could not be separated from the monodisperse QD-labeled antibody bioprobes with a simple method. Thus, purification of QD-based bioprobes is a prerequisite to acquire reliable information by imaging. In previous reports about purification methods of QDs, high-performance size exclusion chromatography (HPSEC) is a very effective method. In this work, HPSEC was used to remove the colloidal impurities in hydrophilic CdSe@ZnS QDs and QD-labeled antibody bioprobes, therefore the resolution between suspended agglomerates and monodisperse QD-labeled antibody bioprobes had been improved by the optimization of HPSEC. The purified hydrophilic QDs covered with carboxyl and QD-labeled antibody bioprobes has exhibit more advantages in the applications.Usually, in order to fabricate QD-based bioprobes, purification is the crucial step to minimize the impurities. Many purification methods such as HPSEC, ultracentrifugation, ultrafiltration, and dialysis etc. have been used to purify QDs. Unlike many other chromatographic methods that rely on the specific interaction between stationary phase and samples, HPSEC, merely based on size exclusion or named "molecular sieving effect" is so powerful and capable of obtaining high sample recovery and retaining the activity of biomolecules to the largest extent for its mild operating conditions. Owing to its advantages, HPSEC has been widely used to separate macromolecules such as proteins, polymers or nano-material by their difference in size. Herein, we demonstrated the application of HPSEC to purify hydrophilic CdSe@ZnS QDs or QD-based bioprobes.In this study, hydrophobic QDs were modified with octylamine grafted polyacrylic acid (OPA) to generate hydrophilic QDs (OPA-QDs), and suspended agglomerates of OPA-QDs and excessive empty OPA colloids were removed by HPSEC. Afterwards, OPA-QDs were PEGylated with amino functionalized PEG for further conjugating with streptavidin (SA) or antibody (IgG) via covalent coupling, generating products, QD-SA or QD-IgG. Since unbound biomolecules and suspended agglomerates of QDs were mixed in the final product after coupling of QDs with proteins, and the suspended agglomerates of QDs could not be removed off from monodispers QD-labeld antibody bioprobes by a simple purification method for limited property differences between them. The suspended agglomerates of QDs is harmful to the colloidal stability of monodisperse QD-based bioprobes, and a factor lead to nonspecific effect in the tumor marker detection using breast cancer tissue slices or in vivo. The separation of conjugates of QDs and functional proteins still could not perfectly achieve because of the limited nature difference between QD-based bioprobes and functional biomolecules. Therefore, it is still a challenge to prepare monodisperse QD-based bioprobes. Undesired byproducts, suspended agglomerates of QDs, excessive SA and IgG had been separated from QD-based bioprobes by HPSEC. At last, the monodisperse QD-based bioprobes were used for fluorescence imaging of tumor markers using breast cancer tissue slices. Tumor marker molecule mapping with the QD-based bioprobes was specific, and nonspecific cellular binding could be minimized. These results demonstrate that the fabricated QD-based bioprobes were of excellent properties on the basis of purification by HPSEC, which is promising in the nanobiodetection field. Although the CdSe@ZnS QDs is an "old member" of QDs, it is suitable to be used to establish a HPSEC-coupled purification process which is a potential standard purified operation to other nanoparticals because of its outstanding repeatability and high recovery. Under premise of optimization progress such as a suitable concentration of sodium chloride had been added into the mobile phase, flow rate of mobile phase had been reduced, multiple chromatographic columns had been connected with polythene pipes to increase the column length. The base line separation of suspended agglomerates of QDs and monodisperse QD-labeled antibody bioprobes had been achieved.HPSEC was used for purification of hydrophilic CdSe@ZnS QDs and CdSe@ZnS QD-based bioprobes. The main contents in this dissertation are as follows:1. Hydrophilic octylamine grafted polyacrylic acid modified QDs (OPA-QDs) were prepared, and purified by HPSEC to remove excess OPA and suspended agglomerates of OPA-QDs. Purified monodisperse CdSe@ZnS OPA-QDs possess excellent colloidal stability for at least one year. Then, purified CdSe@ZnS OPA-QDs were PEGylated with amino-terminated polyethylene glycol (NH2-PEG-NH2) to prepare PEGylated QDs with an amino terminal.2. Subsequently, PEGylated QDs were conjugated to streptavidin (SA) or second antibody (Immunoglobulin G, IgG), generating QD-SA and QD-IgG, respectively. The unbound functional molecules and suspended agglomerates were removed from the fresh prepared QD-based bioprobes via HPSEC, and the monodisperse QD-SA and QD-IgG were used to detect biomarkers (high molecular weight cytokeratin (CK34?E12), E-cadherine (E-cad) and p120 catenin (p120)) on slices of human breast tumor tissue. The purified bioprobes showed specific positive signals without nonspecific adsorption. Thus, the HPSEC-coupled system proposed in the current work is potent and universal for the generation of purified and monodisperse QD-based bioprobes, which are of great prospects in biomedicine. The molecular mapping of tumor markers in the breast cancer tissue slices is consistent with the image stained by immunohistochemistry (IHC) which is a traditional method.3. The PEGylated CdSe@ZnS QDs coupled with thiol modificated anti-human epidermal growth factor receptor 2 antibody developed in mouse (anti-HER2 Ab), the QD-labeled anti-HER2 antibody bioprobes (QD-HER2-Ab) was purified by HPSEC. The unbound antibody and suspended agglomerates of QDs were removed from the fresh prepared QD-HER2-Ab, and the pure and homogeneous granularity QD-HER2-Ab were used as tumor target bioprobes in vivo. The QD-HER2-Ab do not cause apparent acute toxicities in mice during a week post a signal intravenous injection with a dosage of 15 nmol·kg-1 related to the mouse body weight, and it shows high tumor targeting ability in a breast tumor xenograft nude mouse model (at 24 h time point). The concentration of residual cadmium (Cd) in livers which were injected with HPSEC purified QD-HER2-Ab and that of unpurified QD-HER2-Ab were 8.3 ng/kg and 56.7 ng/kg related to liver weight, respectively. The purified QD-HER2-Ab do not cause apparent symptoms of inflammation in main visceral organs of administrated mice, the blood biochemical indicators of kidney or liver are all in normal levels. And the background of imaging is minimized for the reduction of residual QDs in peripheral blood and liver, the background of immunofluorescence imaging and the acute toxicity significant decrease in the same time, so the specificity was improved. The HPSEC purified QD-HER2-Ab provides an accurate tool for tumor target imaging and HER2 detection in vitro and in vivo, and providing a basis for purification of other QD-based bioprobes.In addition, the conjugates of PEGylated QDs and anti-alpha fetoprotein (AFP) antibody (QD-AFP-Ab) was prepared and purified by HPSEC, and the HPSEC purified QD-AFP-Ab was used in tumor targeting of the liver tumor xenograft nude mouse model. LM9 cell-line was chosen as in vitro cell culture models, and LM9 cells were used to produce tumor under nude mouse subcutaneous. Tumor target imaging in the nude mice which was bearing the liver tumor had been effectively achieved by HPSEC purified QD-AFP-Ab based on in vivo detection.In conclusion, this work is mainly focused on the purification of hydrophilic CdSe@ZnS QDs and CdSe@ZnS QD-based bioprobe, and HPSEC was used as the main purified method. The results show that the purified monodisepers QD-based bioprobes have conspicuous superiority in tumor marker determination in vitro and in vivo detection.