Immunofluorescent labeling of cancer marker Her2 and other cellular targets with semiconductor quantum dots (original) (raw)

Immunofluorescent Labeling of Tissues and Free Cells via QD-Protein Conjugates

Quantum dots (QDs), semiconductor inorganic nanocrystals, have a big importance in immunofluorescent labeling. We have prepared water soluble QDs in the size range from 2.8 to 4.4 nm with 3-mercaptopropionic acid (MPA) as a hydrophilic surface ligand. To reduce their cytotoxicity, QDs are coated by two epitaxial layers of CdS and ZnS. For the immunofluorescent labeling we used cadmium telluride QDs of size 3.7 nm with maximum emission wavelength at 610 nm conjugated with various proteins, antibody to membrane CD3 protein, antibody to proliferating cell nuclear antigen and annexin V. From a variety of techniques described in literature, we have chosen the conjugation using the zero-length cross-linkers such as 1-ethyl-3-(3-dimethyl-3-aminopropyl) carbodiimide hydrochloride and N-hydroxysulfosuccinimide, which bind molecules via carboxyl and primary amino groups. This method is compared with the so called oriented conjugation method where an amine group of modified QDs reacts with oxi...

Biofunctional quantum dots as fluorescence probe for cell-specific targeting

Colloids and Surfaces B: Biointerfaces, 2014

We describe here the synthesis, characterization, bioconjugation, and application of water-soluble thioglycolic acid TGA-capped CdTe/CdS quantum dots (TGA-QDs) for targeted cellular imaging. Antihuman epidermal growth factor receptor 2 (HER2) antibodies were conjugated to TGA-QDs to target HER2-overexpressing cancer cells. TGA-QDs and TGA-QDs/anti-HER2 bioconjugates were characterized by fluorescence and UV-Vis spectroscopy, X-ray diffraction (XRD), hydrodynamic sizing, electron microscopy, and gel electrophoresis. TGA-QDs and TGA-QDs/anti-HER2 were incubated with cells to examine cytotoxicity, targeting efficiency, and cellular localization. The cytotoxicity of particles was measured using an MTT assay and the no observable adverse effect concentration (NOAEC), 50% inhibitory concentration (IC 50 ), and total lethal concentration (TLC) were calculated. To evaluate localization and targeting efficiency of TGA-QDs with or without antibodies, fluorescence microscopy and flow cytometry were performed. Our results indicate that antibody-conjugated TGA-QDs are well-suited for targeted cellular imaging studies.

Exploiting the UV excited size-dependent emission of PDMS-coated CdTe quantum dots for in vitro simultaneous multicolor imaging of HepG2 cellular organelles

Multicolor bioimaging can be referred to as the imaging method that non-invasively visualizes biological processes using fluorophores. Over the years, this technique has been primarily used in the areas of diagnostic and theranostic applications such as the detection of pathogens, identification of diseasespecific markers, and cancer detection. Fluorophores such as organic dyes are widely used in bioimaging studies. However, organic dyes exhibit a major limitation of excitation and emission spectral overlap, especially when multispectral bioimaging is considered. Quantum dots (QDs), in contrast, hold great potential due to their properties such as size-tunable narrow emission, single excitation in UV, photo and chemical stability, high fluorescence lifetime, simple surface modification process, etc. Thus, QDs can be a good alternative to traditional fluorophores for bioimaging applications. Herein, the previously reported poly dimethyl siloxane (PDMS)-coated CdTe QDs (PQDs) are used to simultaneously image cellular organelles such as lysosomes, mitochondria, nucleus, and actin of HepG2. Briefly, blue, green, yellow, and orange PQDs are conjugated to CD68 ab. (Lysosomes), mitochondria ab., nuclear antigen ab. and smooth muscle actin ab. respectively using EDC-NHS chemistry. The intracellular organelle targeting of the conjugated QDs is assessed by colocalization with the commercially available dyes. Finally, PQD-conjugates are used to simultaneously image four cellular organelles of HepG2 at an excitation wavelength of 405 nm. The present study demonstrates the potential of PQDs as a fluorophore in simultaneous multicolor bioimaging.

Advanced procedures for labeling of antibodies with quantum dots

Analytical Biochemistry, 2011

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms.

Use of quantum dots for live cell imaging

Nature Methods, 2004

Monitoring interactions within and among cells as they grow and differentiate is a key to understanding organismal development. Fluorescence microscopy is among the most widely used approaches for highresolution, noninvasive imaging of live organisms 1,2 , and organic fluorophores are the most commonly used tags for fluorescence-based imaging 3. Despite their considerable advantages in live cell imaging, organic fluorophores are subject to certain limitations. Fluorescent quantum dots (QDs) are inorganic fluorescent nanocrystals that overcome many of these limitations and provide a useful alternative for studies that require long-term and multicolor imaging of cellular and molecular interactions 4,5. For labeling specific cellular proteins, QDs must be conjugated to biomolecules that provide binding specificity. Bioconjugation approaches vary with the surface properties of the hydrophilic QD used. The mixed surface self-assembly approach is recommended for conjugating biomolecules to QDs capped with negatively charged dihydroxylipoic acid (DHLA) (Fig. 1). In this approach DHLA-capped QDs are conjugated to proteins using positively charged adaptors 6-8 , for example, a naturally charged protein (e.g., avidin 9), a protein fused to positively charged leucine zipper peptide (zb) 8 or a protein fused to pentahistidine peptide (5× His) 10. The use of avidin permits stable conjugation of the QDs to ligands, antibodies or other molecules that can be biotinylated, whereas the use of proteins fused to a positively charged peptide or oligohistidine peptide obviates the need for biotinylating the target molecule. This procedure describes the bioconjugation of QDs and specific labeling of both intracellular and cell-surface proteins 4,5. For generalized cellular labeling, QDs not conjugated to a specific biomolecule may be used; various strategies are presented in Box 1, Generalized Labeling of Live Cells. MATERIALS REAGENTS Quantum dots (QDs; e.g., Quantum Dot Corporation or Evident Technology) Cells or tissue for labeling, prepared appropriately depending on the application (QDs can be used to tag live cells, label cell-surface proteins, or label fixed cells or tissue sections) Amylose resin (New England Biolabs) Antibodies of interest Avidin (Sigma Chemicals) Bovine serum albumin (BSA), 1% in PBS Lipid-based transfection reagent (e.g., Lipofectamine 2000 (Invitrogen) or Fugene 6 (Roche)) Maltose (Sigma Chemicals) Maltose-binding protein fused to the basic leucine zipper domain (MBP-zb) and protein G fused to the basic leucine zipper domain (PG-zb) expressed and purified from bacteria as described elsewhere 7 .

In Vivo Tumor-Targeted Fluorescence Imaging Using Near-Infrared Non-Cadmium Quantum Dots

Bioconjugate Chemistry, 2010

This article reported the high tumor targeting efficacy of RGD peptide labeled near-infrared (NIR) non-cadmium quantum dots (QDs). After using poly(ethylene glycol) to encapsulate InAs/InP/ZnSe QDs (emission maximum at about 800 nm), QD800-PEG dispersed well in PBS buffer with the hydrodynamic diameter (HD) of 15.9 nm and the circulation half-life of ∼29 min. After coupling QD800-PEG with arginine-glycine-aspartic acid (RGD) or arginine-alanine-aspartic acid (RAD) peptides, we used nude mice bearing subcutaneous U87MG tumor as models to test tumor-targeted fluorescence imaging. The results indicated that the tumor uptake of QD800-RGD is much higher than those of QD800-PEG and QD800-RAD. The semiquantitative analysis of the region of interest (ROI) showed a high tumor uptake of 10.7 (1.5%ID/g in mice injected with QD800-RGD, while the tumor uptakes of QD800-PEG and QD800-RAD were 2.9 (0.3%ID/g and 4.0 (0.5%ID/g, respectively, indicating the specific tumor targeting of QD800-RGD. The high reproducibility of bioconjunction between QDs and the RGD peptide and the feasibility of QD-RGD bioconjugates as tumor-targeted fluorescence probes warrant the successful application of QDs for in vivo molecular imaging.

Ultra Q-bodies: quench-based antibody probes that utilize dye-dye interactions with enhanced antigen-dependent fluorescence

Scientific Reports, 2014

Recently, we described a novel reagentless fluorescent biosensor strategy named Quenchbody, which functions via the antigen-dependent removal of the quenching effect on a fluorophore that is attached to a single-chain antibody variable region. To explore the practical utility of Quenchbodies, we prepared antibody Fab fragments that were fluorolabeled at either one or two of the N-terminal regions, using a cell-free translation-mediated position-specific protein labeling system. Unexpectedly, the Fab fragment labeled at the heavy chain N-terminal region demonstrated a deeper quenching and antigen-dependent release compared to that observed using scFv. Moreover, when the Fab was fluorolabeled at the two N-termini with either the same dye or with two different dyes, an improved response due to enhanced quenching via dye-dye interactions was observed. On the basis of this approach, several targets, including peptides, proteins, and haptens, as well as narcotics, were quantified with a higher response up to 50-fold. In addition, differentiation of osteosarcoma to osteoblasts was successfully imaged using a similarly fluorolabeled recombinant Fab protein prepared from E. coli. Due to its versatility, this ''Ultra-Quenchbody'' is expected to exhibit a range of applications from in vitro diagnostics to the live imaging of various targets in situ. P rotein-based fluorescent probes are widely used for the specific detection of a trace amount of substances in vivo and in vitro. However, to date, most of these developed probes (biosensors) rely on the function of a target-specific natural receptor, and each construct must be designed and empirically and individually tested for its performance, which is a laborious and time-consuming process. As a possible solution for this issue, we recently reported a general strategy for the detection of a broad range of biomolecules using a fluorolabeled antibody fragment called ''Quenchbody (Q-body)'' 1. The fluorescence of Qbody (a position-specific fluorolabeled single-chain variable region, scFv) was quenched in its antigen-free state due to the interaction (photoinduced electron transfer, PET) of the intramolecular tryptophan (Trp) residues to the attached dye, i.e., TAMRA. However, once Q-body binds to its target antigen, the quenching interaction is released by an antigen-induced conformational change, resulting in increased fluorescence of the exposed dye from the antibody. This increase in fluorescence is rapid and reflects the target antigen concentration. Unlike other fluorescence-based probes, including reagentless biosensors 2-4 , Q-bodies may be generated for a range of targets, from small molecules such as narcotics to larger proteins, such as serum albumins, on the basis of this assay principle. However, despite these merits, some Q-bodies exhibit a limited fluorescence response and/or sensitivity, which is most likely due to insufficient Trp-mediated quenching of the attached dye in the absence of antigen. Moreover, the stability and affinity of scFv, compared with its parental full-size antibody or fragment of antigen binding (Fab), is of controversial concern 5,6. To resolve this problem, we propose a new strategy to generate Fab-based Qbodies, which incorporates the use of multiple dyes. Our results clearly demonstrate its superior performance compared to scFv-based Q-bodies in both detection sensitivity and response. Furthermore, as an application to