Labeling Agent Research Articles

Monolayer single crystal two-dimensional quantum dots via ultrathin cutting and exfoliating

Two-dimensional (2D) atomically thin quantum dots (QDs) possess extraordinary electrical and optical properties. However, fabricating high quality 2D QDs via a universal and reliable technique remains a challenge. Here, we report a simple strategy to prepare high quality, monolayer single crystal 2D QDs via ultrathin cutting 2D bulk single crystals by ultramicrotome, followed by an exfoliation process. The as-prepared 2D QDs have pristine surface, high quality, high monolayer yield and high photoluminescence quantum yield (the highest photoluminescence quantum yield of WS2 is 18%), which can be used as promising, low toxic, biocompatible, and good cell-permeability fluorescent labeling agents for in vitro imaging.

Synthesis and Structural Optimization of Iridium(III) Solvent Complex for Electrochemiluminescence Labeling of Histidine-Rich Protein and Immunoassay Applications for CRP Detection.

The reaction between an iridium(III) solvent complex and the histidine site of biomolecules as one kind of novel bioconjugation approaches has received much attention during the past few years. To extend this novel bioconjugation approach into electrochemiluminescence (ECL) immunoassay and optimize the performances, three iridium(III) solvent complexes with different C∧N bidentate main ligands have been designed and synthesized in this work. Bovine serum albumin (BSA) as the standard histidine-rich protein is initially employed to evaluate the labeling performances by comparing the ECL intensity of the same amount of BSA labeled by different iridium(III) solvent complexes. Importantly, a magnetic beads-based sandwich immunoassay platform using Ir-dmpq (iridium(III) acetonitrile complex with 2-(3,5-dimethylphenyl)quinoline as the main ligand) as a structurally optimized labeling agent has been successfully constructed to detect C-reactive protein (CRP, an important biomarker of systemic inflammation in clinic), and the limit of detection based on this novel labeling agent could reach below 1 ng/mL, which may further pave the way for applications of the iridium(III) solvent complex in histidine-rich protein ECL labeling beyond fluorescence labeling.

Bovine serum albumin determination based on methylene blue detection by photothermal lens spectroscopy

We report on a new sensitive method, for bovine serum albumin quantification, which is based on the use of methylene blue as a labelling agent combined with photothermal lens detection. In the presence of sodium dodecyl sulfate, methylene blue forms a dimer which can react with bovine serum albumin producing dedimerization. We found that, the photothermal signal decreases proportional to the concentration of bovine serum albumin added to the system composed by dodecyl sulfate and methylene blue. Therefore, the change of the signal intensity is linearly proportional to the concentration of bovine serum albumin.Under the optimized analytical conditions, used in this work, the photothermal signal is linearly dependent on the concentration of bovine serum albumin in the range of 0.5 × 10−6–7.5 × 10−5 gmL−1 with a regression coefficient R2 = 0.9954. The relative standard deviation for BSA determination at 3.5 × 10−5 gmL−1 (n = 5) is 2.3% and the achieved detection limit is 3.5 × 10−7 gmL−1.

Beyond chimerism analysis: methods for tracking a new generation of cell-based medicines.

The analysis of chimerism is crucial to determine the status of patients receiving hematopoietic stem cell transplantation. The variety of relevant techniques available today range from those that analyse nucleic acids (i.e. polymerase chain reaction based, next generation sequencing) and cellular phenotype (i.e. flow cytometry) to sophisticated imaging (particularly multimodal imaging using labelling agents). However, current developments of advanced therapies bring chimerism studies into a new dimension in which methods for detection of donor cells in the patient need to adapt to a wider range of cell- and gene-based medicines, routes of administration, target organs and pathologies. Herein we describe and analyze the toolkit of suitable labelling and detection methodologies with actual examples along with a discussion on challenges ahead and potential solutions. Remarkably, existing methods commonly used in chimerism analysis are suitable for use with new cell- and gene-based medicines. Indeed, new developments may facilitate the evolution and combination of such methodologies to the use of non-invasive and highly informative approaches.

Pharmacological and Cellular Significance of Triazole-Surrogated Compounds

: Heterocyclic compounds have been at the hierarchy position in academia, and industrial arena, particularly the compounds containing triazole-core are found to be potent with a broad range of biological activities. The resistance of triazole ring towards chemical (acid and base) hydrolysis, oxidative and reductive reaction conditions, metabolic degradation and its higher aromatic stabilization energy makes it a better heterocyclic core as therapeutic agents. These triazole-linked compounds are used for clinical purposes for antifungal, anti-mycobacterium, anticancer, anti-migraine and antidepressant drugs. Triazole scaffolds are also found to act as a spacer for the sake of covalent attachment of the high molecular weight bio-macromolecules with an experimental building blocks to explore structure-function relationships. Herein, several methods and strategies for the synthesis of compounds with 1,2,3-triazole moiety exploring Hüisgen, Meldal and Sharpless 1,3-dipolar cycloaddition reaction between azide and alkyne derivatives have been deliberated for a series of representative compounds. Moreover, this review article highlights in-depth applications of the [3+2]-cycloaddition reaction for the advances of triazole-containing antibacterial as well as metabolic labelling agents for the in vitro and in vivo studies on cellular level.

Signal to Noise Ratio as a Cross-Platform Metric for Intraoperative Fluorescence Imaging.

Real-time molecular imaging to guide curative cancer surgeries is critical to ensure removal of all tumor cells; however, visualization of microscopic tumor foci remains challenging. Wide variation in both imager instrumentation and molecular labeling agents demands a common metric conveying the ability of a system to identify tumor cells. Microscopic disease, comprised of a small number of tumor cells, has a signal on par with the background, making the use of signal (or tumor) to background ratio inapplicable in this critical regime. Therefore, a metric that incorporates the ability to subtract out background, evaluating the signal itself relative to the sources of uncertainty, or noise is required. Here we introduce the signal to noise ratio (SNR) to characterize the ultimate sensitivity of an imaging system and optimize factors such as pixel size. Variation in the background (noise) is due to electronic sources, optical sources, and spatial sources (heterogeneity in tumor marker expression, fluorophore binding, and diffusion). Here, we investigate the impact of these noise sources and ways to limit its effect on SNR. We use empirical tumor and noise measurements to procedurally generate tumor images and run a Monte Carlo simulation of microscopic disease imaging to optimize parameters such as pixel size.

Nanoscale magnetic imaging enabled by nitrogen vacancy centres in nanodiamonds labelled by iron-oxide nanoparticles.

Nanodiamonds containing the nitrogen vacancy centre (NV) have a significant role in biosensing, bioimaging, drug delivery, and as biomarkers in fluorescence imaging, due to their photo-stability and biocompatibility. The optical read out of the NV unpaired electron spin has been used in diamond magnetometry to image living cells and magnetically labelled cells. Diamond magnetometry is mostly based on the use of bulk diamond with a large concentration of NV centres in a wide field fluorescence microscope equipped with microwave excitation. It is possible to correlate the fluorescence maps with the magnetic field maps of magnetically labelled cells with diffraction limit resolution. Nanodiamonds have not as yet been implemented to image magnetic fields within complex biological systems at the nanometre scale. Here we demonstrate the suitability of nanodiamonds to correlate the fluorescence map with the magnetic imaging map of magnetically labelled cells. Nanoscale optical images with 17 nm resolution of nanodiamonds labelling fixed cells bound to iron oxide magnetic nanoparticles are demonstrated by using a single molecule localisation microscope. Nanoscale magnetic field images of the magnetised magnetic nanoparticles spatially assigned to individual cells are superresolved by the NV centres within nanodiamonds conjugated with the magnetic nanoparticles with 20 nm resolutions. Our method offers a new platform for the super-resolution of optical magnetic imaging in biological samples conjugated with nanodiamonds and iron-oxide magnetic nanoparticles.

Synthesis of Horseradish Peroxidase-Gold Nanoparticle Conjugate through Green Route

Synthesis of horseradish peroxidase-gold nanoparticle conjugates (HRP-AuNPs) has been studied for the development of biofunctionalized gold nanoparticles (AuNPs) through biogenic route. Herein, horseradish peroxidase enzyme has been used to synthesize gold nanoparticles at room temperature in tricine buffer. The morphology and size distribution of HRP-AuNPs conjugates were obtained by different techniques including dynamic light scattering (DLS), UV-visible (UV-vis) spectrophotometry, scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDX). The enzyme activity of HRP-AuNP conjugate was compared with free enzyme to determine their catalytic efficiency. The results suggests that HRP-AuNP conjugates are monodisperse particles with average hydrodynamic diameter of 83.93 ± 2.1 nm, zeta potential of about -18.4 ± 1.1 mV and higher enzyme activity towards H2O2 as compared to free horseradish peroxidase. These biofunctionalized gold nanoparticles could act as tag or labeling agent for various applications.

Live Cell Imaging with Holotomography and Fluorescence

Abstract

A chemical covalent tactic for bio-thiol sensing and protein labeling agent design.

Discovering novel chemical reactions is important for bioanalysis. Herein, we report a tactic for bio-thiol sensing and protein labeling agent design by the installation of a sulfoxide group onto the skeleton of various fluorophores, and powerfully validate its abilities, which may shed light on the development of specific protein tags to give insight into their biological functions.

Sulfenic Acid-Directed Chemical Probes and Workflows for Assessing Protein Oxidation

Cyanine based chemosensing platforms have successfully been employed over the past couple of decades in various fields of biomolecular sciences. Still a substantial number of recent advances and improvements on this class of compounds are reported in the art. This paper presents our latest work on the improved synthetic approach, study on photophysical properties, and biosensing applicability of monomethine cyanine dyes. The series of mono-, di- and tricationic dyes showed up to 5-fold enhanced resistance against photobleaching compared to the commercially available Thiazole Orange (TO). The title compounds were studied as potential molecular probes for the detection of deoxyribonucleic acid, demonstrating their capacity as excellent fluorescent labeling agents. Depending on the dye chemical structure, current Cl-TO compounds exhibit up to 834-fold enhanced fluorescence emission and form stable complexes with Calf Thymus-DNA. The calculated binding constants were found to be higher than several conventional fluorogenic dyes for nucleic acid detection. All studied derivatives appeared as less cytotoxic than the Thiazole Orange. IC50 concentrations in human fibroblasts MRC5 cell line were calculated up to 50 μM for the synthesized Cl-TO dyes, and 0.5 μM for the parental Thiazole Orange. Two of the dyes were found very competent in post-electrophoretic visualization of DNA. As demonstrated by the agarose gel electrophoresis, the staining efficiency and detection limits of the dyes were comparable to the widely used Ethidium Bromide. The tricationic dye revealed great potential for cell cycle analysis in G1, S and G2 phases. The chlorinated TO derivatives readily stain human cells in vivo, while they can effectively be applied for eukaryotic and microbial cell staining.

Synthesis and evaluation of a radioiodinated BODIPY derivative as a thiol-labeling agent.

Labeling agents with radioisotopes or fluorescent dyes are useful for investigating the biodistributions of biologically active proteins and peptides. Compared with molecular imaging with a single modality, dual imaging probes provide complementary information for each modality. The development of a dual radioisotope/fluorescence agent for protein labeling would thus be valuable for both preclinical and clinical applications. In this study, we designed and synthesized a radioiodinated BODIPY derivative (BODIPY-ML) with a maleimide group as a thiol-labeling agent. In the presence of N-chlorosuccinimide and 1% acetic acid, [125 I]BODIPY-ML was successfully obtained at a radiochemical yield of 42%. In conjugation studies, model proteins including RGD peptides and anti-HER2 VHH were successfully labeled with BODIPY-ML via covalent bonds. The results demonstrated the feasibility of the radioiodinated BODIPY as a dual-labeling agent via thiol groups.

Covalently modified carboxyl side chains on cell surface leads to a novel method toward topology analysis of transmembrane proteins

The research on transmembrane proteins (TMPs) is quite widespread due to their biological importance. Unfortunately, only a little amount of structural data is available of TMPs. Since technical difficulties arise during their high-resolution structure determination, bioinformatics and other experimental approaches are widely used to characterize their low-resolution structure, namely topology. Experimental and computational methods alone are still limited to determine TMP topology, but their combination becomes significant for the production of reliable structural data. By applying amino acid specific membrane-impermeable labelling agents, it is possible to identify the accessible surface of TMPs. Depending on the residue-specific modifications, new extracellular topology data is gathered, allowing the identification of more extracellular segments for TMPs. A new method has been developed for the experimental analysis of TMPs: covalent modification of the carboxyl groups on the accessible cell surface, followed by the isolation and digestion of these proteins. The labelled peptide fragments and their exact modification sites are identified by nanoLC-MS/MS. The determined peptides are mapped to the primary sequences of TMPs and the labelled sites are utilised as extracellular constraints in topology predictions that contribute to the refined low-resolution structure data of these proteins.

Ptychographic modulation engine: a low-cost DIY microscope add-on for coherent super-resolution imaging

Imaging of biological cells and tissues often relies on fluorescent labels, which offer high contrast with molecular specificity. The use of exogenous labeling agents, however, may alter the normal physiology of the bio-specimens. Complementary to the established fluorescence microscopy, label-free quantitative phase imaging provides an objective morphological measurement tool for bio-specimens and is free of variability introduced by contrast agents. Here, we report a simple and low-cost microscope add-on, termed ptychographic modulation engine (PME), for super-resolution quantitative phase imaging. In this microscope add-on module, we attach a diffuser to a 3D-printed holder that can be mechanically moved to different x-y positions. We then use two vibrational motors to introduce random positional shifts to the diffuser. The add-on module can be placed between the objective lens and the specimen in most existing microscope platforms. Thanks to the diffuser modulation process, the otherwise inaccessible high-resolution object information can now be encoded into the captured images. In the ptychographic phase retrieval process, we jointly recover the complex object wavefront, the complex diffuser profile, and the unknown positional shifts of the diffuser. We demonstrate a four-fold resolution gain over the diffraction limit of the employed 2× objective lens. We also test our approach for in vitro cell imaging, where we are able to adjust the focus after the data has been captured. The reported microscope add-on provides a turnkey solution for super-resolution quantitative phase imaging. It may find applications in label-free bio-imaging where both large field-of-view and high resolution are needed.

Silica-Coated Magnetic Nanoparticles Decrease Human Bone Marrow-Derived Mesenchymal Stem Cell Migratory Activity by Reducing Membrane Fluidity and Impairing Focal Adhesion.

For stem cell-based therapies, the fate and distribution of stem cells should be traced using non-invasive or histological methods and a nanomaterial-based labelling agent. However, evaluation of the biophysical effects and related biological functions of nanomaterials in stem cells remains challenging. Here, we aimed to investigate the biophysical effects of nanomaterials on stem cells, including those on membrane fluidity, using total internal reflection fluorescence microscopy, and traction force, using micropillars of human bone marrow-derived mesenchymal stem cells (hBM-MSCs) labelled with silica-coated magnetic nanoparticles incorporating rhodamine B isothiocyanate (MNPs@SiO2(RITC)). Furthermore, to evaluate the biological functions related to these biophysical changes, we assessed the cell viability, reactive oxygen species (ROS) generation, intracellular cytoskeleton, and the migratory activity of MNPs@SiO2(RITC)-treated hBM-MSCs. Compared to that in the control, cell viability decreased by 10% and intracellular ROS increased by 2-fold due to the induction of 20% higher peroxidized lipid in hBM-MSCs treated with 1.0 µg/µL MNPs@SiO2(RITC). Membrane fluidity was reduced by MNPs@SiO2(RITC)-induced lipid oxidation in a concentration-dependent manner. In addition, cell shrinkage with abnormal formation of focal adhesions and ~30% decreased total traction force were observed in cells treated with 1.0 µg/µL MNPs@SiO2(RITC) without specific interaction between MNPs@SiO2(RITC) and cytoskeletal proteins. Furthermore, the migratory activity of hBM-MSCs, which was highly related to membrane fluidity and cytoskeletal abnormality, decreased significantly after MNPs@SiO2(RITC) treatment. These observations indicated that the migratory activity of hBM-MSCs was impaired by MNPs@SiO2(RITC) treatment due to changes in stem-cell biophysical properties and related biological functions, highlighting the important mechanisms via which nanoparticles impair migration of hBM-MSCs. Our findings indicate that nanoparticles used for stem cell trafficking or clinical applications should be labelled using optimal nanoparticle concentrations to preserve hBM-MSC migratory activity and ensure successful outcomes following stem cell localisation.

Synthesis of symetrical near-infrared dyes containing benzo[c,d]indole system

Five symmetrical near infrared dyes having different linker between the two heterocycles were synthesized by condensation of N-alkylbenzo[c,d]indolium salts with either squaric acid or imine. All synthesized dyes showed strong absorbance in the near-infrared region of 850-1015 nm and can be used as nucleic acid detection or fluorescent labeling agents for proteins. Three of the dyes 21, 22 and 25 containing functionalized N-substituents (ester group) can be further modified or covalently attached to target. The structures of these cyanine dyes where characterized by virtue of NMR, UV, MALDI and Elemental analysis.Keywords: Synthesis, cyanine, Polymethine dyes, Near-IR dyes, Absorption, Chromophore.

Fast and sensitive delineation of brain tumor with clinically compatible moxifloxacin labeling and confocal microscopy.

Delineation of brain tumor margins during surgery is critical to maximize tumor removal while preserving normal brain tissue to obtain optimal clinical outcomes. Although various imaging methods have been developed, they have limitations to be used in clinical practice. We developed a high-speed cellular imaging method by using clinically compatible moxifloxacin and confocal microscopy for sensitive brain tumor detection and delineation. Moxifloxacin is a Food and Drug Administration (FDA) approved antibiotic and was used as a cell labeling agent through topical administration. Its strong fluorescence at short visible excitation wavelengths allowed video-rate cellular imaging. Moxifloxacin-based confocal microscopy (MBCM) was characterized in normal mouse brain specimens and visualized their cytoarchitecture clearly. Then, MBCM was applied to both brain tumor murine models and two malignant human brain tumors of glioblastoma and metastatic cancer. MBCM detected tumors in all the specimens by visualizing dense and irregular cell distributions, and tumor margins were easily delineated based on the cytoarchitecture. An image analysis method was developed for automated detection and delineation. MBCM demonstrated sensitive delineation of brain tumors through cytoarchitecture visualization and would have potentials for human applications, such as a surgery-guiding method for tumor removal.

Biosynthesized Gold Nanoparticles: In Vivo Study of Near-Infrared Fluorescence (NIR)-Based Bio-imaging and Cell Labeling Applications.

Near infrared (NIR) fluorescence imaging is a striking imaging modality for biomedical and clinical applications due to its deep tissue penetration and low phototoxicity. The major issue with NIR dyes is their non-specific distribution and requirement of tagging with biomolecules for specific tissue localization. Till now, there have been no imaging agents available that can distribute into a specific organ without the need for targeted ligands, which remains as an unmet clinical need. In the present study, we demonstrate that the Zinnia elegans plant extract (abbreviated as ZE) assisted synthesis of highly biocompatible gold nanoparticles (AuZE), leading to their non-invasive bio-imaging applications in the NIR region (red at 820 nm emission: NIR region). AuZE and ZE both exhibited green fluorescence at 350 nm excitation and red fluorescence in the NIR region (710 nm). We verified the source of this fluorescence, which originates from the fluorescent molecules present in the ZE extract. After intraperitoneal administration in C57BL6 mice, very interestingly, AuZE is distributed into the brain of C57BL6 mice without the need for any targeted ligand and exhibited bright red fluorescence in the NIR region (710 nm excitation, 820 nm emission) as evidenced by non-invasive imaging as well as ICPOES techniques. We further explored the activity of ZE and AuZE as cell labeling agents (B16F10 cells were pre-incubated with AuZE and implanted into mice, and the fluorescence was monitored), which could be applicable for graft transplantation biology. To the best of our knowledge, this is the first report that demonstrates the versatile applications of green synthesized gold nanoparticles using a ZE extract. Considering these exciting results and fruitful outcomes, the ZE and AuZE NPs would stand as an alternative imaging agent to commercially available NIR dyes and change the conventional fluorescence-based bio-imaging strategies. Therefore, the biosynthesized AuNPs open new directions for future research to explore these latest observations in the field of disease diagnosis and therapy.

Moxifloxacin Labeling-Based Multiphoton Microscopy of Skin Cancers in Asians.

Although multiphoton microscopy (MPM) can visualize both cell and extracellular matrix (ECM) structures of the skin in high-contrast without exogenous labeling, label-free MPM is usually too slow to image clinically relevant large regions. A high-speed MPM method would be beneficial for evaluating clinical skin specimens by increasing the imaging area. In this study, moxifloxacin labeling-based MPM (moxifloxacin MPM) was characterized in various human skin cancer specimens. Moxifloxacin ophthalmic solution was used for cell-labeling and MPM imaging was conducted afterwards. Moxifloxacin MPM was characterized in ex vivo normal human skin and skin cancer specimens in comparison with the label-free MPM and fluorescence confocal microscopy (FCM) using acridine orange as a labeling agent. Then, moxifloxacin MPM was applied to various ex vivo human skin cancer specimens including basal cell carcinoma (BCC), squamous cell carcinoma (SCC), dermatofibrosarcoma protuberans (DFSP). Results of moxifloxacin MPM were compared with bright-field clinical and histopathologic findings. Moxifloxacin MPM imaged both cells and collagen in the skin, similarly to label-free MPM, but with enhanced fluorescence intensities in cells and enhanced imaging speeds. Moxifloxacin MPM imaged cells in the skin similarly to acridine orange-based FCM. Moxifloxacin MPM of various human skin cancer specimens imaged their specific cellular features. The microscopic features detected in moxifloxacin MPM were confirmed with histological images. This observational pilot study demonstrated that moxifloxacin MPM could detect specific cellular features of various skin cancers in good correlation with histopathological images in Asian patients at the higher imaging speed than label-free MPM.Lasers Surg. Med. © 2019 Wiley Periodicals, Inc.

Fluorine-19 Cellular MRI Detection of In Vivo Dendritic Cell Migration and Subsequent Induction of Tumor Antigen-Specific Immunotherapeutic Response.

A major hurdle in the advancement of cell-based cancer immunotherapies is the inability to track in vivo therapeutic cell migration. With respect to dendritic cell (DC)-based cancer immunotherapies, this lack of knowledge represents an even greater hurdle as the quantity of tumor-antigenspecific DC reaching a secondary lymphoid organ post injection is predictive of the magnitude of the ensuing tumor-specific immune response. We propose fluorine-19 (F-19) cellular magnetic resonance imaging (MRI) as a suitable and non-invasive imaging modality capable of detecting and quantifying DC migration in vivo and thus, serving as a surrogate marker of DC-based immunotherapeutic effectiveness. Murine DC were generated from bone marrow precursors and labeled with a [19F]perfluorocarbon ([19F]PFC)-based cell labeling agent. DC were characterized by viability and phenotyping assessments as well as characterized by ability to induce in vivo tumor-specific immune responses following immunization in a B16-F10 mouse model of melanoma. The in vivo migration of [19F]PFC (PFC)-labeled DC was first compared to control unlabeled DCby microscopyand then measured using F-19 cellular MRI. Culture conditions were optimized such that > 90% of DC labeled with PFC without affecting viability, phenotype, and function. This optimization permitted consistent detection of PFC-labeled DC migration using F-19 cellular MRI and resulted in the first successful comparison of in vivo migration between PFC-labeled and control unlabeled therapeutic cells of the same origin. PFC-labeled DC are migration-competent in vivo in a B16-F10 tumor-bearing mouse model. We report a non-invasive and longitudinal imaging modality capable of detecting and quantifying therapeutic cell migration at both 9.4 and 3Tesla (T) and suitable for therapeutic cell tracking in a tumor-bearing mouse model. F-19 MRI cell tracking is broadly applicable across disease states and is conducive to clinical translation.