Design, Synthesis, and Use of MMP-2 Inhibitor-Conjugated Quantum Dots in Functional Biochemical Assays.

The development of chemically designed matrix metalloprotease (MMP) inhibitors has advanced the understanding of the roles of MMPs in different diseases. Most MMP probes designed are fluorogenic substrates, often suffering from photo- and chemical instability and providing a fluorescence signal of moderate intensity, which is difficult to detect and analyze when dealing with crude biological samples. Here, an inhibitor that inhibits MMP-2 more selectively than Galardin has been synthesized and used for enzyme labeling and detection of the MMP-2 activity. A complete MMP-2 recognition complex consisting of a biotinylated MMP inhibitor tagged with the streptavidin-quantum dot (QD) conjugate has been prepared. This recognition complex, which is characterized by a narrow fluorescence emission spectrum, long fluorescence lifetime, and negligible photobleaching, has been demonstrated to specifically detect MMP-2 in in vitro sandwich-type biochemical assays with sensitivities orders of magnitude higher than those of the existing gold standards employing organic dyes. The approach developed can be used for specific in vitro visualization and testing of MMP-2 in cells and tissues with sensitivities significantly exceeding those of the best existing fluorogenic techniques.

Dynamic changes in chromatin conformation at the TNF transcription start site in T helper lymphocyte subsets.

Tumor necrosis factor (TNF) is one of the key primary response genes in the immune system that can be activated by a variety of stimuli. Previous analysis of chromatin accessibility to DNaseI demonstrated open chromatin conformation of the TNF proximal promoter in T cells. Here, using chromatin probing with restriction enzyme EcoNI and micrococcal nuclease we show that in contrast to the proximal promoter, the TNF transcription start site remains in a closed chromatin configuration in primary T helper (Th) cells, but acquires an open state after activation or polarization under Th1 and Th17 conditions. We further demonstrate that transcription factor c-Jun plays a pivotal role in the maintenance of open chromatin conformation at the transcription start site of the TNF gene.

Detection of carcinoembryonic antigen using single-domain or full-size antibodies stained with quantum dot conjugates.

Compact single-domain antibodies (sdAbs) are nearly 13 times smaller than full-size monoclonal antibodies (mAbs) and have a number of advantages for biotechnological applications, such as small size, high specificity, solubility, stability, and great refolding capacity. Carcinoembryonic antigen (CEA) is a tumor-associated glycoprotein expressed in a variety of cancers. Detection of CEA on the tumor cell surface may be carried out using anti-CEA antibodies and conventional fluorescent dyes. Semiconductor quantum dots (QDs) are brighter and more photostable than organic dyes; they provide the possibility for labeling of different recognition molecules with QDs of different colors but excitable with the same wavelength of excitation. In this study, the abilities for specific detection of CEA expressed by tumor cells with anti-CEA sdAbs biotinylated in vitro and in vivo, as well as with anti-CEA mAbs biotinylated in vitro, were compared using flow cytometry and the conjugates of streptavidin with QDs (SA-QDs). The results demonstrated that either in vitro or in vivo biotinylated anti-CEA sdAbs are more sensitive for cell staining compared to biotinylated anti-CEA mAbs. The data also show that simultaneous use of biotinylated sdAbs with highly fluorescent SA-QDs can considerably improve the sensitivity of detection of CEA on tumor cell surfaces.

Quantum dot-based lab-on-a-bead system for multiplexed detection of free and total prostate-specific antigens in clinical human serum samples.

An immunodiagnostic lab-on-a-bead suspension microarray based on microbeads encoded with quantum dots (QDs) has been developed and preclinically validated for multiplexed quantitative detection of prostate cancer markers in human serum samples. The sensitivity and specificity of the microarray are similar to those of "gold-standard" single-analyte ELISA. Moreover, the array has an improved immunoassay capacity, ensures quantitative detection of multiple cancer biomarkers and may be operational in a considerably wider dynamic range of concentrations. The array is characterized by reduced time and cost of analysis and is compatible with classical flow cytometers. Proof-of-concept preclinical tests ensured simultaneous quantitative determination of free and total prostate-specific antigens in human serum, with clear discrimination between the control and clinical samples. The proposed approach is flexible and paves the way to development of a wide variety of immunodiagnostic assays for multiplexed early diagnosis of various diseases. FROM THE CLINICAL EDITOR: Early diagnosis of cancer can result in better prognosis for patients. Thus, the use of specific tumor markers is widely employed in clinical practice. Traditional screening methods only employ single markers. The authors here developed a microarray system based on microbeads encoded with quantum dots (QDs), which can be used for multiplexed quantitative detection. The validated results on patient samples should lead to the development of a wider variety of assays for other diseases.

Single-cell analysis of somatic mutation burden in mammary epithelial cells of pathogenic BRCA1/2 mutation carriers.

Inherited germline mutations in the breast cancer gene 1 (BRCA1) or BRCA2 genes (herein BRCA1/2) greatly increase the risk of breast and ovarian cancer, presumably by elevating somatic mutational errors as a consequence of deficient DNA repair. However, this has never been directly demonstrated by a comprehensive analysis of the somatic mutational landscape of primary, noncancer, mammary epithelial cells of women diagnosed with pathogenic BRCA1/2 germline mutations. Here, we used an accurate, single-cell whole-genome sequencing approach to first show that telomerized primary mammary epithelial cells heterozygous for a highly penetrant BRCA1 variant displayed a robustly elevated mutation frequency as compared with their isogenic control cells. We then demonstrated a small but statistically significant increase in mutation frequency in mammary epithelial cells isolated from the breast of BRCA1/2 mutation carriers as compared with those obtained from age-matched controls with no genetically increased risk for breast cancer.

Quantum-dot-based suspension microarray for multiplex detection of lung cancer markers: preclinical validation and comparison with the Luminex xMAP® system.

A novel suspension multiplex immunoassay for the simultaneous specific detection of lung cancer markers in bronchoalveolar lavage fluid (BALF) clinical samples based on fluorescent microspheres having different size and spectrally encoded with quantum dots (QDEM) was developed. The designed suspension immunoassay was validated for the quantitative detection of three lung cancer markers in BALF samples from 42 lung cancer patients and 10 control subjects. Tumor markers were detected through simultaneous formation of specific immune complexes consisting of a capture molecule, the target antigen, and biotinylated recognition molecule on the surface of the different QDEM in a mixture. The immune complexes were visualized by fluorescently labeled streptavidin and simultaneously analyzed using a flow cytometer. Preclinical validation of the immunoassay was performed and results were compared with those obtained using an alternative 3-plex immunoassay based on Luminex xMAP® technology, developed on classical organic fluorophores. The comparison showed that the QDEM and xMAP® assays yielded almost identical results, with clear discrimination between control and clinical samples. Thus, developed QDEM technology can become a good alternative to xMAP® assays permitting analysis of multiple protein biomarkers using conventional flow cytometers.

Advanced procedure for oriented conjugation of full-size antibodies with quantum dots.

Ideal nanoparticle-based nanoprobes should contain on their surface homogeneously oriented highly active affinity molecules, e.g., antibodies (Abs), and should not exceed 15 nm in diameter. Direct conjugation of quantum dots (QDs) with Abs through cross-linking of QD amines with the sulfhydryl groups resulting from the reduction of the Ab disulfide bonds is a generally accepted technique. However, this procedure yields conjugates where Abs are oriented irregularly. This decreases the number of functionally active Abs on the nanoparticle surface, because some Ab recognition sites face inwards and cannot interact with the target moieties. Here, we describe an advanced procedure of Ab reduction, affinity purification, and QD-Ab conjugation with optimized critical steps. We have developed a method for partially reducing the Abs yielding highly functional 75 kDa heavy-light chain Ab fragments. Affinity purification of these Ab fragments followed by their tagging with QDs results in QD-Ab conjugates with largely improved functionality compared to those obtained according to the standard procedures. The new approach can be extended to conjugation of any type of Abs with different semiconductor, noble metal, or magnetic nanocrystals.

Oriented conjugation of single-domain antibodies and quantum dots.

Nanoparticle-based biodetection routinely employs monoclonal antibodies (mAbs) for targeting. However, the large size of mAbs limits the number of ligands per nanoparticle and severely restricts the bioavailability and distribution of these probes in a sample. Furthermore, conventional conjugation techniques provide nanoprobes with irregular orientation of mAbs on the nanoparticle surface and often provoke mAb unfolding. Here, we describe a protocol for engineering a new generation of ultrasmall diagnostic nanoprobes through oriented conjugation of semiconductor quantum dots (QDs) with 13 kDa single-domain antibodies (sdAbs) derived from llama immunoglobulin G (IgG). The sdAbs are conjugated with QDs in a highly oriented manner via an additional cysteine residue specifically integrated into the sdAb C-terminus. The resultant nanoprobes are <12 nm in diameter, ten times smaller in volume compared to the known alternatives. They have been proved highly efficient in flow cytometry and immunuhistochemical diagnostics. This approach can be easily extended to other semiconductor and plasmonic nanoparticles.