Direct In Situ Hybridization with Oligonucleotide Functionalized Quantum Dot Probes

Abstract

Coming from the material sciences, fluorescent semiconductor nanocrystals, also known as quantum dots (QDs), have emerged as powerful fluorescent probes for a wide range of biological imaging applications. QDs have several advantages over organic dyes which include higher brightness, better resistance to photobleaching, and simplified multicolor target detection. In this chapter, we describe a rapid assay for the direct imaging of multiple repetitive subnuclear genetic sequences using QD-based FISH probes. Streptavidin-coated QDs (SAvQDs) are functionalized with short biotinylated oligonucleotides and used in a single hybridization/detection step. These QD-FISH probes penetrate both intact interphase nuclei and metaphase chromosomes and show good targeting of dense chromatin domains. Importantly, the broad absorption spectra of QDs allows two sequence specific QD-FISH probes of different colors to be simultaneously imaged with a single laser excitation wavelength. This method, which requires minimal custom conjugation, is easily expandable and offers the experimentalist a new alternative to increase flexibility in multicolor cytogenetic FISH applications of repetitive DNAs.

Highly Sensitive Single Domain Antibody–Quantum Dot Conjugates for Detection of HER2 Biomarker in Lung and Breast Cancer Cells

“…16 QDs should be surface functionalized with specific recognition molecules for target detection, 17À22 such as primary or secondary antibody (Ab), 19,22,23 streptavidin, 19,22 peptides, 22,24 proteins, 22,25,26 and oligonucleotides. 22,27,28 Two most common strategies for QDs' surface functionalization with Ab involve either direct cross-linking reaction of carboxylic/amino surface groups of QDs with amino/sulfhydryl groups of Abs or an indirect interaction of streptavidin coated QDs with biotinylated Abs. 13,29 However, both approaches have their own shortcomings.…”

“…Highly fluorescent semiconductor nanocrystals, quantum dots (QDs), with their unique size-dependent physical and chemical properties have been suggested as ideal candidates for this purpose. − The immense number of immunolabeling studies already published demonstrates that QDs are on their way of becoming the preferred fluorescent imaging probes. ,− Their properties, such as broad tunable fluorescence emission spectra that range from ultraviolet (UV) to infrared (IR), high surface to volume ratios and large absorption coefficients across the whole optical spectrum, make them ideal candidates for biomedical imaging, and immunofluorescent staining, especially of fixed cells and tissues . For cancer biomarker detection, QDs should be surface functionalized with specific recognition molecules for target detection, − such as primary or secondary antibody (Ab), ,, streptavidin, , peptides, , proteins, ,, and oligonucleotides. ,, Two most common strategies for QDs’ surface functionalization with Ab involve either direct cross-linking reaction of carboxylic/amino surface groups of QDs with amino/sulfhydryl groups of Abs or an indirect interaction of streptavidin coated QDs with biotinylated Abs. , However, both approaches have their own shortcomings. Inherently, QD surface functionalization with conventional Abs (Figure A), also known as immunoglobulin G (IgG) molecules (molecular weight of around 150 kDa), results in the formation of large conjugates, which are not optimum if intracellular target detection is required and the process of bioconjugation can lead to denaturation of these IgG molecules rendering them inactive .…”

Diagnosis of porcine circovirus type 2 infection with a combination of immunomagnetic beads, single-domain antibody, and fluorescent quantum dot probes

“…Normally, QDbased fluorescent probes are produced by conjugating QDs with recognition molecules. Recent research has shown that QD-based probes can be successfully functionalized with recognition molecules such as peptides [25], antibodies [19,40], or nucleic acids [6,41]. QD-based fluorescent probes have been used in a variety of laboratory applications, such as the detection of cancer cells, bacteria, and viruses [11,13,33].…”

Combining M-FISH and Quantum Dot technology for fast chromosomal assignment of transgenic insertions

“…While QDs have a broad excitation spectrum, the excitation range is the same for each class of QD regardless of its emission wavelength, which makes it possible for them to be simultaneously excited, but distinguished from other fluorophores in a single exposure. Although the use of QD technology in FISH analysis is a relatively new field of investigation, a number of independent research groups have already explored their possible applications to chromosomal studies [ 10 - 14 ]. However, to our knowledge this is the first attempt at combining QD technology with multicolor karyotyping by M-FISH for physical mapping purposes.…”