Bioconjugated quantum dots for multiplexed and quantitative immunohistochemistry

Abstract

Bioconjugated quantum dots (QDs) provide a new class of biological labels for evaluating biomolecular signatures (biomarkers) on intact cells and tissue specimens. In particular, the use of multicolor QD probes in immunohistochemistry is considered one of the most important and clinically relevant applications. At present, however, clinical applications of QD-based immunohistochemistry have achieved only limited success. A major bottleneck is the lack of robust protocols to define the key parameters and steps. Here, we describe our recent experience, preliminary results and detailed protocols for QD-antibody conjugation, tissue specimen preparation, multicolor QD staining, image processing and biomarker quantification. The results demonstrate that bioconjugated QDs can be used for multiplexed profiling of molecular biomarkers, and ultimately for correlation with disease progression and response to therapy. In general, QD bioconjugation is completed within 1 day, and multiplexed molecular profiling takes 1-3 days depending on the number of biomarkers and QD probes used.

Bioconjugation of quantum dots: Review & impact on future application

“…The exposed thiols (hinge region) are sufficiently far away from the hypervariable region for QDbioconjugation to occur [58]. The resultant active fragments of full-size Igs readily interact with maleimide-, pyridyl disulfide-, or iodacetyl-activated QDs to form a covalent bond.…”

“…In comparison with biotin-avidin binding, this ''His-tag'' method has several advantages such as a controlled orientation of the binding ligand (a His-tag can be conveniently fused to proteins and peptides at a particular site), compact overall probe sizes and low production costs. Although the stability of the His-Ni 2+ attachment could be a problem under the harsh conditions of immunohistochemical staining, previous research has shown that the interaction of His 6 with Ni-NTA is virtually unaffected by high ionic strength (up to 1 M), nonionic detergents, organic solvents, ethanol or glycerol up to 30%, reducing agents (bmercaptoethanol) or highly denaturing conditions (urea or guanidine hydrochloride) [58]. This strategy has also been used to develop a QD-probe for performing Western blot analysis with a shorter analysis time and a better sensitivity when compared with the classical Western blot [59].…”

“…The drawback of this conjugation strategy is the degree of uncontrollability of the resulting conjugate, as undesirable protein aggregation can occur through the presence of primary amines on the biomolecules' surface. The procedure discussed above (functionalization with hydrazides) is more selective as conjugation is occurring exclusively on the polysaccharide chain [58].…”

Impact of nanotechnology in breast cancer

“…Yezhelyev and colleagues have developed an assay, using bioconjugated quantum dots, that allows simultaneous quantitative detection of estrogen receptor, progesterone receptor and HER2 (ERBB2) in paraffin-embedded cultured breast cancer cell lines and paraffin-embedded human breast cancer clinical tissue sections [2,6,7]. Quantitative expression of the breast cancer bio markers correlated with conventional immunohistochemical analysis and semi-quantitative western blotting [2,6,7].…”

“…Quantitative expression of the breast cancer bio markers correlated with conventional immunohistochemical analysis and semi-quantitative western blotting [2,6,7].…”