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Synthesis of novel small-molecule fluorescently labeled probes for the in vitro imaging of KCa3.1 channels
摘要： In order to facilitate the in vitro visualization of KCa3.1 channel-expressing cells, novel small-molecule imaging probes were designed and developed. Senicapoc showing high affinity and excellent selectivity towards the KCa3.1 channels was selected as targeting component. Different BODIPY dyes (15 - 20) were synthesized and connected by a Cu-catalyzed azide alkyne [3+2]cycloaddition with propargyl ether derivative 8 of senicapoc yielding fluorescently labeled ligands 21 - 26 targeting KCa3.1 channels. The novel dimethylpyrrole-based imaging probes 25 and 26 allow staining of KCa3.1 ion channels in NSCLC cells following a simple, fast and efficient protocol. The specificity was shown by removing the punctate staining pattern by pre-incubation with senicapoc. The density of KCa3.1 channels detected with fluorescent probe 25 and by immunostaining was identical. The punctate structure of the labeled channels could be observed in living cells as well. Molecular modeling studies showed binding of the senicapoc targeting component towards the binding site within the ion channel and orientation of the linker with the dye along the inner surface of the ion channel.
关键词： labeled ligands,KCa3.1 channel,fluorescent probes,non-small cell lung cancer cells,molecular modelling,imaging agents,cycloaddition
更新于2025-09-23 15:19:57
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[IEEE 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - Munich, Germany (2019.6.23-2019.6.27)] 2019 Conference on Lasers and Electro-Optics Europe & European Quantum Electronics Conference (CLEO/Europe-EQEC) - In Flow Manipulation and Characterization of Cancer Cells by Coherent Computational Microscopy
摘要： Liquid biopsy has shown remarkably promising in oncology for the early diagnosis of cancer through the detection of circulating biomarkers such as circulating tumor cells (CTCs). Recent evidences suggest that CTCs represent effective prognostic and predictive biomarkers to monitor/predict therapy efficacy in breast, colon and prostate cancers [1,2]. However, the frequency of CTCs in blood is approximately 1 to 10 cells per 10 mL of blood, which is as challenging as looking for a needle in a haystack. In microfluidics, Digital Holography (DH) has been shown to be a promising technique to characterize CTCs with the aim to detect them inside a heterogeneous liquid sample. DH is label-free, real-time and gives access to the complex amplitude of the object [3-6]. Thus, any classification approach based on the holographic signature can exploit a reach information content to take a decision. Moreover, the flexible refocusing capability of DH imaging allows to inspect an entire liquid volume with a single capture. This enables the high-throughput inspection of blood and other bodily fluids rapidly flowing inside microfluidic channels. In DH, the sample is probed from one single direction and the phase delay introduced by the sample in through transmission acts as a contrast agent. Hence, the optical thickness measurable by DH imaging is an integral information, i.e. the sum of all the contributions experienced by the coherent light during its passage through the sample. In order to decouple the refractive index from the physical thickness and to resolve its distribution along the optical axis, tomography exploits multiple recordings, probing the sample from different angles and combining the corresponding phase-contrast maps [7,8]. Various schemes have been proposed to minimize the number of sampling angles and to make the recording stage faster in order to match the requirements and time constraints imposed by real biological problems. Here we show the recent advances of in-flow holographic tomography, which exploits a controlled induced rotation of the sample inside the microfluidic channel to probe it from different view angles with no mechanical rotation of the source beam [8,9]. We introduce an effective algorithm to recover from the recorded phase maps the set of angles required as input of the optical projection tomography algorithm [7-9]. We show the application of holographic flow tomography to the characterization of different cancer cells [10], namely breast cancer cells, ovarian cancer cells and neuroblastoma. We also discuss different possibilities of Lab-on-a-Chip design and flow engineering that allow us to induce controlled rotations while maintaining the high-throughput nature of DH microscopy [9,11,12]. In the next future, the large amount of data obtainable by this approach will be used to train a neural network devoted to classify CTCs, distinguishing them from the other components of a blood stream.
关键词： liquid biopsy,cancer cells,microfluidics,circulating tumor cells,holographic tomography,Lab-on-a-Chip,digital holography
更新于2025-09-16 10:30:52
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BRET based dual-colour (visible/near-infrared) molecular imaging using a quantum dot/EGFP–luciferase conjugate
摘要： Owing to its high sensitivity, bioluminescence imaging is an important tool for biosensing and bioimaging in life sciences. Compared to fluorescence imaging, bioluminescence imaging has a superior advantage that the background signals resulting from autofluorescence are almost zero. In addition, bioluminescence imaging can permit long-term observation of living cells because external excitation is not needed, leading to no photobleaching and photocytotoxicity. Although bioluminescence imaging has such superior properties over fluorescence imaging, observation wavelengths in bioluminescence imaging are mostly limited to the visible region. Here we present bioluminescence resonance energy transfer (BRET) based dual-colour (visible/near-infrared) molecular imaging using a quantum dot (QD) and luciferase protein conjugate. This bioluminescent probe is designed to emit green and near-infrared luminescence from enhanced green fluorescent protein (EGFP) and CdSeTe/CdS (core/shell) QDs, where EGFP–Renilla luciferase (RLuc) fused proteins are conjugated to the QDs. Since the EGFP–RLuc fused protein contains an immunoglobulin binding domain (GB1) of protein G, it is possible to prepare a variety of molecular imaging probes functionalized with antibodies (IgG). We show that the BRET-based QD probe can be used for highly sensitive dual-colour (visible/near-infrared) bioluminescence molecular imaging of membrane receptors in cancer cells.
关键词： EGFP,luciferase,bioluminescence imaging,cancer cells,BRET,quantum dot,molecular imaging
更新于2025-09-16 10:30:52
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Graphene Quantum Dots: Efficient Mechanosynthesis, White-Light and Broad Linearly Excitation-Dependent Photoluminescence and Growth Inhibition on Bladder Cancer Cells
摘要： Heteroatom-doped graphene quantum dots (GQDs) have attracted considerable attention due to their potential applications in luminescent materials and biology. In this work, we developed a solvent-free gram-scale mechanochemical method for the preparation of nitrogen-doped graphene quantum dots (N-GQDs) with the highest solubility (31 mg/mL) in water reported to date. Commercial graphite was sheared and cut through grinding with solid melamine, then ground with solid KOH to get sub-5-nm-sized, 1-3-layered N-GQDs. Notably, these N-GQDs exhibit white-light emission and a broad excitation-dependent full-color photoluminescence from 463 nm to 672 nm. When the excitation light ranged from 325 nm to 485 nm, these mechanochemically-obtained N-GQDs exhibited bright white-light emission. Intriguingly, the change of emission wavelength has two-stage linear relationships with the change of the excitation, and the inflection point is at 580 nm (excited at 550 nm). The difference between the emission and excitation wavelength decreases from 138 to 12 nm, which also shows two-stage linear relationships with the change of the excitation wavelength. It is notable that the PL quantum yields of them are high, up to 26.6%. Furthermore, we studied the inhabitation of as-obtained N-GQDs on bladder cancer cells (UMUC-3); as a result, with the increase of the concentration of N-GQDs, the proliferation of cancer cells was obviously prohibited.
关键词： bladder cancer cells,nitrogen-doped,mechanochemical method,photoluminescence,graphene quantum dots
更新于2025-09-16 10:30:52
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Laser-ablation-synthesized nanoparticles and animal cell lines studies
摘要： Nanoparticles (NPs) synthesized by laser ablation in distilled water were used to study their biological effect on normal and cancer cells. Parameters such as cell morphology, cell proliferation and viability were examined for treated cell lines, and the effect was represented in terms of cells cytotoxicity using standard procedures. The study reveals the higher cytotoxic effect of nanoparticles on cancerous cells of breast, melanoma and colon origin compared to normal fibroblast cells NIH-3T3. Furthermore, DNA fragmentation assay results demonstrated the apoptosis mediated cell death in nanoparticle-treated cancer cells. The distinct role of nanoparticles in normal and cancer cells of different origin showed that nanoparticles were specific to cause cytotoxicity in particular cancer cells type. NPs exhibit cytotoxic effects in cancer cells by inducing apoptosis. These studies provide fundamental evidence for the easy, simple and safe mode of nanoparticles synthesis and their application in cancer cells death.
关键词： nanoparticles,Cancer cells,cytotoxicity,laser ablation
更新于2025-09-12 10:27:22
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Chemo-treated 4T1 breast cancer cells radiation response measured by single and multiple cell ionization using infrared laser trap
摘要： We present a study that uses a laser trapping technique for measurement of radiation sensitivity of untreated and chemo-treated cancer cells. We used a human mammary tumor cell line (4T1) treated by an antitumor compound, 2-Dodecyl-6-methoxycyclohexa-2, 5-diene-1,4-dione (DMDD), which was extracted from the root of Averrhoa carambola L. The untreated control group, and both 2-hour and 24-hour treated groups of 4T1 cells were used in this study. The absorbed threshold ionization energy (TIE) and the threshold radiation dose (TRD) were determined using a high-power infrared laser (at 1064 nm) trap by single and multiple cells trapping and ionization. The results were analyzed using descriptive and t-statistics. The relation of the TIE and TRD to the mass of the individual cells were also analyzed for different hours of treatment in comparison with the control group. Both TIE and TRD decrease with increasing treatment periods. However, the TRD decreases with mass regardless of the treatment. Analyses of the TRD for single vs multiple cells ionizations within each group have also consistently showed this same behavior regardless of the treatment. The underlying factors for these observed relations are explained in terms of radiation, hyperthermia, and chemo effects.
关键词： threshold ionization energy,chemo-treated cancer cells,threshold radiation dose,4T1 breast cancer cells,laser trapping technique,DMDD,radiation sensitivity
更新于2025-09-11 14:15:04
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Novel Fluorescent C2-Symmetric Sequential On-Off-On Switch for Cu2+ and Pyrophosphate and Its Application in Monitoring of Endogenous Alkaline Phosphatase Activity
摘要： A doubly armed hydrazone-based FLRHYDDFP probe selectively detects Cu2+ and pyrophosphate (PPi) ions through an colorimetric response-“colorless → yellow → colorless”- as well as “on-off-on” photonic switching response under physiological conditions in a sequential manner. The binding stoichiometries of the analytes Cu2+ and PPi were 1:2 and 2:4 for FLRHYDDFP-Cu2+ and Cu2+/PPi, respectively. The sequential sensing ability of FLRHYDDFP toward Cu2+ and PPi, attributed to effective complexation-aided d→π* electron transfer (ET) from Cu2+ to FLRHYDDFP and intramolecular charge/electron transfer from FLRHYDDFP to FLRHYDDFP+, resulted in the formation of a non-symmetric Cu2+ chelate that provided a yellow-colored solution with a significant bathochromic shift from 376 to 446 nm in the UV-Vis spectrum and quenching in the emission spectrum. Upon addition of PPi, Cu2+ was extruded from the complex, resulting in a revival of the fluorescence centered at 572 nm. Thus, sequential addition of Cu2+ and PPi yielded a colorless–yellow–colorless transition under visible light and on-off-on switching under 365-nm light (fluorescence). The lowest detection limits for Cu2+ and PPi, when using colorimetric and fluorimetric methods, were in the sub-micromolar and nanomolar levels, respectively. By exploiting this PPi sensing strategy, invitro as well as endogenous alkaline phosphatase activity could be monitored effectively, as demonstrated by exploiting the intracellular production or residual PPi in human salivary glands (normal) and cancer cell lines.
关键词： Endogenous Alkaline Phosphatase,C2v Symmetry,d→π* Electron Transfer,Intramolecular Charge/Electron Transfer,Human Salivary Gland Cells and Cancer Cells,Hydrazone
更新于2025-09-11 14:15:04
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X-ray irradiation effects on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells investigated by Raman micro-spectroscopy
摘要： Raman micro-spectroscopy was performed in vitro on nuclear and membrane regions of single SH-SY5Y human neuroblastoma cells after irradiation by graded X-ray doses (2, 4, 6, 8 Gy). The acquired spectra were analyzed by principal component analysis (PCA) and interval-PCA (i-PCA) methods. Biochemical changes occurring in the different regions of single cells as a consequence of the radiation exposure were observed in cells fixed immediately after the irradiation. The most relevant effects arose from the analysis of the spectra from the cell nucleus region. The observed changes were discussed in terms of the modifications in the cell cycle, resulting in an increase in the DNA-related signal, a protein rearrangement and changes in lipid and carbohydrates profiles within the nucleus. Potential markers of an apoptotic process in cell population irradiated with 6 and 8-Gy X-ray doses could have been singled out. No significant effects were found in spectra from cells fixed 24 h after the irradiation, thus suggesting the occurrence of repairing processes of the X-ray induced damage.
关键词： X-ray effects on DNA, lipids, proteins and carbohydrates,Single SH-SY5Y human cancer cells,Raman micro-spectroscopy,Cellular nucleus and membrane,Multivariate analysis
更新于2025-09-11 14:15:04
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Single-Cell Imaging of Metastatic Potential of Cancer Cells
摘要： Molecular imaging of metastatic "potential" is an unvanquished challenge. To engineer biosensors that can detect and measure the metastatic "potential" of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin remodelers required for cell migration, which are enriched in cancers but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates prometastatic signaling by multiple oncogenic receptors, we named them "integrators of metastatic potential (IMP)." IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady state, detected those few cells that have acquired the highest metastatic potential, and tracked their enrichment during metastasis. These findings provide proof of concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way.
关键词： GIV/Girdin,cancer cells,phosphoproteome,FRET biosensors,metastatic potential
更新于2025-09-10 09:29:36
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EXTRACELLULAR AND INTRACELLULAR SYNTHESIS OF SILVER NANOPARTICLES
摘要： The cellular synthesis of nanoparticle is a green process and alternative for a conventional process for the preparation of silver nanoparticles. In our research, focus has been given to the development of an efficient and eco-friendly viable process for the synthesis of silver nanoparticles using cancer and non-cancerous cells, a cell culture that was isolated. The results of this investigation are observed that silver nanoparticles could be induced to synthesis intra- and extra-cellularly using mammalian cells such as cancerous and non-cancerous cells. The silver nanoparticles are synthesized by the cancer and non-cancerous cells such as HeLa (Homo sapiens, human), SiHa, and human embryonic kidney-293 cell lines. The silver nanoparticles were characterized by ultraviolet (UV)-visible spectroscopy, transmission electron microscopy (TEM), and X-ray powder diffraction (XRD). The silver nanoparticles exhibited maximum absorbance at 415 nm in UV-visible spectroscopy. The XRD confirms the characteristic of the crystal lattice of silver nanoparticles by observing three peaks: Peak at 38 is due to reflection from (111), peak at 44 is due to reflection from (200), and peak at 65 is due to reflection from (220). TEM images showed the formation of stable silver nanoparticles in the cell lines. The method of extraction of intracellular/extracellular synthesis of silver nanoparticles was inexpensive, simple, and effective in large scale with no need to use of complex process equipment. The cancer cell considered as a biological factory at nanoscale dimension which continued to grow after synthesis of silver nanoparticles. The silver reduction by these cancer cells has occurred through energy-dependent processes that lead to the high output of this reaction. Hence, this new approach of using a mammalian cell for the successful synthesis of nanosized silvers could be easily scaled up, which establishes its commercial viability and also useful in the drug delivery and drug targeting.
关键词： Cancer cells,Biosynthesis and characteristics of silver nanoparticles,Silver nanoparticles
更新于2025-09-09 09:28:46