Living HepG2 Cells Research Articles

Robust Fluorescent Nanoprobe for Rapid Evaluation of the Selenium Supplementation Effect and Imaging.

At present, an increasing number of people pay more attention to selenium-enriched food, but the quality of the selenium-enriched food varies. Therefore, there is an urgent need to develop a new tool to assess the effects of selenium supplementation in foods by rapidly detecting the levels of the metabolite selenium selenocysteine (Sec). In this work, a fluorescent nanoprobe CS-Sec was designed, synthesized, and characterized for Sec detection and imaging in living biosystems, which exhibited the advantages of good biocompatibility, excellent water solubility, high sensitivity, high selectivity, and rapid response (2.5 min) for Sec detection and imaging in vitro and in vivo and evaluation of selenium supplementation in selenium-rich foods. Specifically, CS-Sec was constructed by grafting alkyne groups on organic small-molecule fluorescent probes with azide groups on azido chitosan by click chemistry. A 2,4-dinitrophenyl ether (DNB) with a strong intramolecular charge transfer (ICT) effect was selected as a response group and fluorescence-quenching group, which had excellent chemical specificity toward Sec. In addition, CS-Sec has high selectivity and sensitivity toward Sec over other analytes, and an excellent limit of detection (LOD) is as low as 15 nM. Impressively, CS-Sec has been successfully used to detect and image the concentration of Sec in living HepG2 cells and mouse models with exciting results, indicating that the newly constructed CS-Sec can provide a robust molecule tool for the rapid evaluation of the selenium supplementation effect and imaging in the future.

A new benzil-based chemosensor for colorimetric and fluorometric detection of Cu2+ with high selectivity and sensitivity in living cells

A novel benzil-based probe DBBQ containing two julolidine Schiff base arms was synthesized and demonstrated for its highly selective and sensitive detection of Cu2+ ions in CH3CN: HEPES buffers (3:2, v/v, pH=7.4). Upon the addition of Cu2+ ions, the absorption of the probe exhibited remarkable change in color from yellow to brown, which could be easily observed by the naked eyes. In addition, probe DBBQ displayed highly selective fluorescence quenching signal for Cu2+ ions over a wide range of competitive substances. The fluorescence intensity of DBBQ in CH3CN: HEPES buffer (3:2, v/v, pH=7.4) solution showed a good linear response relationship to Cu2+ ions in the range of 0–2.5 μΜ and low detection limit of 0.41 μM. The binding ratio between DBBQ and Cu2+ ions was determined to be 1:1. Probe DBBQ could be potentially used as a chemosensor for Cu2+ ions in living HepG2 cells.

Near-infrared fluorescent probe for ultrasensitive detection of organophosphorus pesticides and visualization of their interaction with butyrylcholinesterase in living cells

The toxicity of organophosphorus pesticides (OPs) can catastrophically cause liver cell damage and inhibit the catalytic activity of cholinesterase. We designed and synthesized a near-infrared fluorescent probe HP-LZB with large Stokes shift which can specifically identify and detect butyrylcholinesterase (BChE) and visually explore the interaction between OPs and endogenous BChE in living cells. Fluorescence was turned on when HP-LZB was hydrolyzed into HP-LZ in the presence of BChE, and OPs could inhibit BChE's activity resulting in a decrease of fluorescence. Six OPs including three oxon pesticides (paraoxon, chlorpyrifos oxon and diazoxon) and their corresponding thion pesticides (parathion, chlorpyrifos and diazinon) were investigated. Both in vitro and cell experiments indicated that only oxon pesticides could inhibit BChE's activity. The limits of detection (LODs) of paraoxon, chlorpyrifos oxon and diazoxon were as low as 0.295, 0.007 and 0.011 ng mL−1 respectively and the recovery of OPs residue in vegetable samples was satisfactory. Thion pesticides themselves could hardly inhibit the activity of BChE and are only toxic when they are converted to their corresponding oxon form in the metabolic process. However, in this work, thion pesticides were found not be oxidized into their oxon forms in living HepG2 cells due to the lack of cytochrome P450 in hepatoma HepG2 cell lines. Therefore, this probe has great application potential in effectively monitoring OPs in real plant samples and visually exploring the interaction between OPs and BChE in living cells.

Pyrazolone compounds could inhibit CES1 and ameliorates fat accumulation during adipocyte differentiation

Carboxylesterase 1 (CES1), a member of the serine hydrolase superfamily, is involved in a wide range of xenobiotic and endogenous substances metabolic reactions in mammals. The inhibition of CES1 could not only alter the metabolism and disposition of related drugs, but also be benefit for treatment of metabolic disorders, such as obesity and fatty liver disease. In the present study, we aim to develop potential inhibitors of CES1 and reveal the preferred inhibitor structure from a series of synthetic pyrazolones (compounds 1–27). By in vitro high-throughput screening method, we found compounds 25 and 27 had non-competitive inhibition on CES1-mediated N-alkylated d-luciferin methyl ester (NLMe) hydrolysis, while compound 26 competitively inhibited CES1-mediated NLMe hydrolysis. Additionally, Compounds 25, 26 and 27 can inhibit CES1-mediated fluorescent probe hydrolysis in live HepG2 cells with effect. Besides, compounds 25, 26 and 27 could effectively inhibit the accumulation of lipid droplets in mouse adipocytes cells. These data not only provided study basis for the design of newly CES1 inhibitors. The present study not only provided the basis for the development of lead compounds for novel CES1 inhibitors with better performance, but also offered a new direction for the explore of candidate compounds for the treatment of hyperlipidemia and related diseases.

A triphenylamine-benzofuran-derived fluorescent probe for monitoring sulfite in Chinese medicinal materials and bioimaging

In this work, a triphenylamine-benzofuran-derived fluorescent probe TBSF was developed for monitoring the sulfite level in Chinese medicinal materials and imaging in living cells. In the testing system, under 445 nm excitation, TBSF responded to sulfite steadily with a 540 nm fluorescence reporting signal. The testing system showed advantages including high sensitivity, rapid response, and high selectivity. In particular, TBSF achieved the sulfite detection in the water decoction of Chinese medicinal materials from both addition and excessive fumigation. It also realized the intracellular imaging of both exogenous and endogenous sulfite in living HepG2 cells. The imaging in water decoction-treated cells inferred the potential for the interdisciplinary detection.

Electrochemical biosensing platform based on AuNWs/rGO-CMC-PEDOT:PSS composite for the detection of superoxide anion released from living cells

Detection of superoxide anion (O2·-) levels holds significant importance for the diagnosis and even clinical treatments of oxidative stress-related diseases. Herein, we prepared a composite electrode material to encapsulate copper-zinc superoxide dismutase (SOD1) for biosensing of O2·-. The sensing material consists of gold nanowires (AuNWs), reduced graphene oxide (rGO), carboxymethyl cellulose (CMC) and PEDOT:PSS. CMC provides abundant -COOH to bind SOD1, with a high adsorption coverage of 1.499 × 10−9 mol cm−2 on the sensor surface. rGO and PEDOT endow the composite with significant conductivity, whereas PSS has antifouling capability. Moreover, AuNWs exhibit excellent electrical conductivity and a high aspect ratio, which promotes electron transfer, and ultimately enhances the catalytic performance of the enzyme. Meanwhile, SOD1(Cu2+) catalyzes the dismutation of O2·- to O2 and H2O2, and H2O2 is then electrochemically oxidized to generate amperometric signals for determination of O2·-. The sensor demonstrates outstanding detection performance for O2·- with a low detection limit of 2.52 nM, and two dynamic ranges (14.30 nM–1.34 μM and 1.34 μM–42.97 μM) with corresponding sensitivity of 0.479 and 0.052 μA μM−1cm−2, respectively. Additionally, the calculated apparent Michaelis constant (Kmapp) of 1.804 μM for SOD1 demonstrates the outstanding catalytic activity and the surface-immobilized enzyme's substrate affinity. Furthermore, the sensor shows the capability to dynamically detect the level of O2·- released from living HepG2 cells. This study provides an inovative design to obtain a biocompatible electrochemical sensing platform with plenty of immobilization sites for biomolecules, large surface area, high conductivity and flexibility.

Neo-construction of a SO2-tunable near-infrared ratiometric fluorescent probe for high-fidelity diagnosis and evaluation hazards of Cd2+-induced liver injury

Cadmium-contaminated water and food are seriously hazardous to the human health, especially liver injury. To understand the entanglement relationship between cadmium ion (Cd2+)-induced liver injury and the biomarker sulfur dioxide (SO2), a reliable bioanalytical tool is urgently needed, detecting SO2 to diagnose and evaluate the extent of liver injury in vivo. Herein, based on the Förster resonance energy transfer (FRET) mechanism, a novel SO2-tunable NIR ratiometric fluorescent probe (SMP) was developed, it was used to diagnose and treat liver injury induced by Cd2+ in biosystems. Specifically, it was constructed by conjugating a NIR dicyanoisophorone with a NIR benzopyranate as the donor and acceptor, respectively, and the ratiometric response of SO2- regulated by the Michael addition reaction. In addition, SMP exhibits rapid reaction time (<15 s), two well-resolved emission peaks (68 nm) with less cross-talk between channels for high imaging resolution, superior selectivity, and low limit of detection (LOD=80.3 nM) for SO2 detection. Impressively, SMP has been successfully used for intracellular ratiometric imaging of Cd2+-induced SO2 and diagnostic and therapeutic evaluation in liver injury mice models with satisfactory results. Therefore, SMP may provide a powerful molecular tool for revealing the occurrence and development relationship between SO2 and Cd2+-induced liver injury. Environmental ImplicationCadmium ions are one of the well-known toxic environmental pollutants, which are enriched in the human body through inhalation of cadmium-contaminated air or from the food chain, leading to damage in various organs, especially liver injury. Therefore, we developed a novel fluorescent probe that can specifically detect SO2 in Cd2+-induced liver injury, which is critically important for the diagnosis and evaluation of Cd2+-induced liver injury diseases. The specific detection of SO2 of this probe has been successfully demonstrated in live HepG2 cells and Cd2+-induced liver injury mice.

Synthesis of a metal-organic framework Cu-Mi-UiO-66-based fluorescent nanoprobe for the simultaneous sensing and intracellular imaging of GSH and ATP.

This study reports a fluorescent nanoprobe operated in fluorescence turn-on mode for simultaneously sensing and imaging intracellular GSH and ATP. By using maleimide-derivatives as the ligand, the bimetallic nanoscale metal-organic framework (NMOF) Cu-Mi-UiO-66 has been synthesized for the first time using a straightforward one-step solvothermal approach, serving as a GSH recognition moiety. Subsequently, a Cy5-labeled ATP aptamer was assembled onto Cu-Mi-UiO-66 via strong coordination between phosphate and zirconium, π-π stacking and electrostatic adsorption to develop the dual-responsive fluorescence nanoprobe Cu-Mi-UiO-66/aptamer. Due to the photoinduced electron transfer (PET) effect between maleimide groups and the benzene ring of the ligand and the charge transfer between Cy5 and the Zr(IV)/Cu(II) bimetal center of the NMOF, the Cu-Mi-UiO-66/aptamer exhibits a fluorescence turn-off status. The Michael addition reaction between the thiol group of GSH and the maleimide on the NMOF skeleton results in turning on of the blue fluorescence of Cu-Mi-UiO-66. Meanwhile, upon specific interaction with ATP, the aptamer changes into internal loop structures and detaches from Cu-Mi-UiO-66, resulting in turning on of the red fluorescence of Cy5. The nanoprobe demonstrated an excellent sensing performance with a good linear range (GSH, 5.0-450.0 μM; ATP, 1.0-50.0 μM) and a low detection limit (GSH, 2.17 μM; ATP, 0.635 μM). More importantly, the Cu-Mi-UiO-66/aptamer exhibits good performance for tracing intracellular concentration variations of GSH and ATP in living HepG2 cells under different stimulations. This study highlights the potential of NMOFs for multiplexed analysis and provides a valuable tool for tumor microenvironment research and early cancer diagnosis.

Real-time monitoring of glucose metabolism and effects of metformin on HepG2 cells using 13C in-cell NMR spectroscopy

Metformin is currently a strong candidate antitumor agent for multiple cancers, and has the potential to inhibit cancer cell viability, growth, and proliferation. Metabolic reprogramming is a critical feature of cancer cells. However, the effects of metformin which targets glucose metabolism on HepG2 cancer cells remain unclear. In this study, to explore the effects of metformin on glucose metabolism in HepG2 cells, we conducted real-time metabolomic monitoring of live HepG2 cells treated with metformin using 13C in-cell NMR spectroscopy. Metabolic tracing with U–13C6-glucose revealed that metformin significantly increased the production of 13C-G3P and 13C-glycerol, which were reported to attenuate liver cancer development, but decreased the production of potential oncogenesis-supportive metabolites, including 13C-lactate, 13C-alanine, 13C-glycine, and 13C-glutamate. Moreover, the expression levels of enzymes associated with the measured metabolites were carried out. The results showed that the levels of ALT1, MCT4, GPD2 and MPC1 were greatly reduced, which were consistent with the changes of measured metabolites in 13C in-cell NMR spectroscopy. Overall, our approach directly provides fundamental insights into the effects of metformin on glucose metabolism in live HepG2 cells, and highlights the potential mechanism of metformin, including the increase in production of G3P and glycerol derived from glucose, as well as the inhibition of glucose incorporation into lactate, alanine, glutamate, and glycine.

An ortho-activation strategy to develop NIR fluorescent probe for rapid imaging of biothiols in vivo

Biothiols are the main antioxidants in regulating the redox balance and resisting oxidative stress in various biological processes, but the long detection time of current fluorescent probes hinders their rapid imaging in vitro and in vivo. To reveal the influx of biothiols, we rationally develop an ortho-activation approach to accelerate the reaction between the probe and biothiols, by introducing electron-withdrawing fluorine atom into the ortho-site of the phenolic hydroxyl group in the NIR probe to generate an ortho-inductive effect. The ortho-fluorine helps to increase the chemical reactivity of the molecular structure, resulting in a significantly shorter detection time (within 5 min) as compared to previous reports (> 20 min for acrylates-based probes in aqueous solution). Based on this approach, our near-infrared probe 2F-RBX can sensitively and efficiently detect endogenous biothiols in living HepG2 cells and in vivo. These data suggest that ortho-activation is a simple and flexible approach to construct sensitive fluorescent probes for rapid imaging of biothiols, and perhaps other molecules in future, under biological circumstances.

Targeting the Mutant Region in K-RAS by RNA Interference Controlling Cell Proliferation and Programmed Cell Death in Liver Cancer Cells.

Background Hepatocellular carcinoma (HCC) is a high-incidence and significant cause of cancer globally. HCC is the world's third most common death-related cancer and the sixth most common tumor. In HCC, various cellular signaling is activated to ensure malignancy transformation, angiogenesis, and metastasis. The most efficient signaling pathway in cancer is mitogen-activated protein kinase (MAPK), which controls malignancy and regulates apoptosis. The mutant k-ras gene led to the activation of the RAS protein with GTPase activity, which stimulated hepatocellular proliferation and transformation. Here we aim to investigate the correlation between the knock-down of k-ras and HCC evaluation in-vitro. &#x0D; Material and Methods We used the HepG2 cell line to investigate the direct connection between the k-ras expression profile and RAF/MEK pathway using a respective siRNA antagonist k-ras. &#x0D; Results Interestingly, the siRNA antagonist k-ras altered cell morphology and a number of the living HepG2 cells. In addition, the transfection of HepG2 cells with the firstly designed siRNA successfully reduced the expression profile k-ras and the relative gene expression of Raf-1 and Mek1, the downstream targets of RAS signaling. Furthermore, interleukin 8 (IL-8) and interleukin 6 (IL-6) were monitored in the fluids media at different time points following transfection. Both IL-6 and Il-8 production significantly increased in cells transfected with siRNA targeting k-ras. &#x0D; Conclusion Our findings provide evidence for the influential role of the k-ras mutated gene in HCC development and suggest the possible regulation of this gene as a potential treatment for HCC.

A new turn on coumarin-based fluorescence probe for Cr3+ detection in aqueous solution

Isatin-3-(7′-Methoxychromone-3′-methylidene) hydrazone (L) was synthesized based on chromone schiff base, and used to construct a novel sensor to detect Cr3+. Fluorescence detection experiments were carried out for a range of different concentrations of Cr3+ in aqueous solutions. A concentration calculation model was built on the basis of eliminating interference of excitation spectrum in the fluorescence spectra with mathematical method. Results showed that probe L displayed a 70-fold fluorescence enhancement upon the addition of Cr3+ due to the photo-induced electron transfer (PET) effect. On the other hand, metal ions except Cr3+ did not cause significant change in either the absorption or the fluorescence spectrum of L. In addition, L showed a good selectivity to Cr3+ over other metal cations, especially Al3+ and Cu2+. The probe L can detect Cr3+ highly and selectively by the direct chelation enhanced fluorescence with a detection limit of 3.14 × 10−6 M. Furthermore, benefiting from their good water solubility and biocompatibility, cell imaging and real-time monitoring of Cr3+ in living HepG2 cells were successfully achieved.

Photoelectrochemical detection of superoxide anions released from mitochondria in HepG2 cells based on the synergistic effect of MnO2@Co3O4 core-shell p-n heterojunction

The detection and comparison of the amount of superoxide anion (O2.-) released by different complexes in mitochondrial electron transport chain (ETC) can locate the main electron leakage sites in mitochondria. In order to realize this, we designed an ultrasensitive photoelectrochemical (PEC) sensor by in situ hydrothermal growth of MnO2 nanosheets on Co3O4 nanowires array modified Ti substrate (NWA|Ti). Due to the formation of a core-shell p-n heterojunction with high specific surface area, tight surface contact and plentiful oxygen vacancies (OVs), MnO2@Co3O4 NWA|Ti possesses a strong visible light absorption, high charges transfer and separation ability. The proposed PEC sensor exhibited a wide linear range of 0.1–50000 nM and a low detection limit of 0.025 nM towards H2O2. Due to the rapid conversion of O2.- to H2O2 inside mitochondria, the PEC sensor can indirectly monitor the electron leakage in the ETC. Specifically, four selected mitochondrial inhibitors specifically inhibited the corresponding complex in mitochondria extracted from living HepG2 cells (hepatocellular carcinoma cells), and the H2O2 levels converted from O2.- was measured by the PEC sensor. It is evident that IQ (ubiquinone binding) site of complex I and Qo (ubiquinol oxidation) site of complex III are the key sites at which electron leakage occurred. This study could provide meaningful information for the diagnosis and treatment of certain disease caused by oxidative stress due to the electron leakage.

Discovery of seven-membered ring berberine analogues as highly potent and specific hCES2A inhibitors

Human carboxylesterase 2A (hCES2A) is a key serine hydrolase responsible for the metabolic clearance of large number of compounds bearing the ester- or amide-bond(s). Inhibition of hCES2A can relieve the chemotherapy-induced toxicity and alter the pharmacokinetic bahaviors of some orally administrate esters-containing agents. However, most of the hCES2A inhibitors show poor cell-membrane permeability and poor specificity. Herein, guided by the structure activity relationships (SAR) of fifteen natural alkaloids against hCES2A, fifteen new seven-membered ring berberine analogues were designed and synthesized, and their anti-hCES2A activities were evaluated. Among all tested compounds, compound 28 showed potent anti-hCES2A effect (IC50 = 1.66 μM) and excellent selectivity over hCES1A (IC50 > 100 μM). The SAR analysis revealed that the seven-membered ring of these berberine analogues was a crucial moiety for hCES2A inhibition, while the secondary amine group of the ring-C is important for improving their specificity over other serine hydrolases. Inhibition kinetic analyses and molecular dynamic simulation demonstrated that 28 strongly inhibited hCES2A in a mixed-inhibition manner, with an estimated Ki value of 1.035 μM. Moreover, 28 could inhibit intracellular hCES2A in living HepG2 cells and exhibited suitable metabolic stability. Collectively, the SAR of seven-membered ring berberine analogues as hCES2A inhibitors were studied, while compound 28 acted as a promising candidate for developing highly selective hCES2A inhibitors.

A mitochondria-targeted ratiometric NIR fluorescent probe for simultaneously monitoring viscosity and ONOO− based on two different channels in living HepG2 cells

Mitochondrial viscosity and ONOO− are involved in various pathological and physiological processes. Abnormal viscosity or irregular ONOO− concentration would disrupt the normal function of cells. Hence, we skillfully designed and synthesized a dual-responsive probe Mito-HC-TZ for the simultaneous detection of ONOO− and viscosity by different channels. 3-ethylbenzothiazolium was acted as fluorescent rotor to regulate the twisted intramolecular charge transfer (TICT) process; the C = C of 3-ethylbenzothiazolium linked to the xanthene core could be easily broken by ONOO−. Thus, the probe responded to viscosity and ONOO− by red and orange fluorescence channels, respectively. Mito-HC-TZ had high selectivity, low cytotoxicity and wide range of pH values. The results of cell imaging experiments showed the probe could be used to respectively monitor the viscosity and ONOO− level in HepG2 cells.

Construction of a ratiometric two-photon ER-targeting fluorescent probe for the imaging of peroxynitrite in living systems

Peroxynitrite (ONOO-) could deplete ER-associated Ca2+ and cause ER stress, and thereby precise and in situ detection of ONOO- at ER is highly important for the study of its biological roles. Herein, we present a ratiometric two-photon ER-targeting fluorescent probe (NAB) for the detection of ONOO- in living cells and brain tissues. NAB utilized 4-bromomethylphenyl borate as a recognition site for ONOO-, and utilized p-toluenesulfonamide as ER-targeting group. After the response of NAB to ONOO-, the 4-bromomethylphenyl borate converted to hydroxyl, resulting in the change of the fluorescence color from blue to yellow. The probe NAB could show highly sensitive response to ONOO- within 6 s, and exhibited excellent selectivity to ONOO-. The probe NAB mainly located at ER and showed excellent ER-targeting property. Ratiometric two-photon imaging demonstrated that the probe NAB could selectively detect exogenous ONOO- at ER, and also could be applied to image metformin-induced ONOO- in living HepG2 cells. Furthermore, NAB can effectively detect the changes of ONOO- level in brain tissues, and revealed that the endogenous ONOO- level at ER showed increase in hippocampus of the depressive mouse.

Activated Two-Photon Near-Infrared Ratiometric Fluorescent Nanoprobe for ONOO- Detection and Early Diagnosis and Assessment of Liver Injury.

Liver injury poses a serious threat to human health and growing evidence suggests that it is closely associated with a biomarker (peroxynitrite, ONOO-). Therefore, considering that the relationship of ONOO- levels with the occurrence and development of liver injury disease remains a challenge, an urgent need exists to develop a reliable and robust tool for its visual rapid diagnosis and assessment. Herein, a two-photon near-infrared (TP-NIR) ratiometric fluorescent nanoprobe (NTC) based on a fluorescence resonance energy transfer (FRET) strategy was designed, synthesized, and characterized, which had the advantages of good water solubility, low background interference, deep tissue penetration, and high imaging resolution. Specially, NTC was constructed by self-assembly of an alkynyl group of a small-molecule fluorescent probe (NR) via click chemistry grafting onto azide chitosan (natural polymeric nanomaterial). NR contained acceptor 1 (NIR fluorophore) and donor 3 (D-π-A structure of naphthalimide derivative fluorophore) with outstanding TP properties that could be activated by ONOO- for the ratiometric detection of ONOO-. Furthermore, in the presence of ONOO-, NTC exhibited a short response time (∼10 s) and high selectivity and sensitivity toward ONOO- with an excellent detection limit as low as 15.3 nM over other reactive oxygen/nitrogen species. Notably, NTC has been successfully employed for ONOO- detection and imaging in living HepG2 cells, liver injury mice tissues, and mice models with satisfactory results. Thus, the construction of this NTC nanoprobe can provide a robust molecule tool for enabling early diagnosis and assessment of liver injury in the future.

A fluorescein-2-(Pyridin-2-ylmethoxy) benzaldehyde conjugate for fluorogenic turn-ON recognition of Hg2+ in water and living cells with logic gate and memory device applications

An effective Hg2+ specific probe, fluorescein-2-(Pyridin-2-ylmethoxy)-benzaldehyde conjugate (FO511), was designed, synthesized and characterized by various instrumental techniques. The sensing behavior of FO511 was investigated by fluorescence technique which clearly established the high selectivity towards Hg2+ through OFF–ON fluorescence response in the presence of other metal ions like Zn2+, Mg2+, Co2+, Ni2+, Cu2+, Pb2+, Al3+, Cr3+, Cd2+, Pd2+, Fe2+ and Fe3+ in aqueous medium (pH 7.2, 10 mM HEPES buffer). The sensing mechanism could be attributed to the Hg2+ triggered spirolactam ring opening of the fluorescein moiety and simultaneous formation of a 1:1 FO511–Hg2+ complex. The interaction and formation of FO511–Hg2+ species was supported by the observations gained from fluorescence titrations, Job’s plot, 1H NMR and HRMS, and other spectroscopic studies. For the Hg2+ interaction towards FO511 the binding constant was evaluated to be (3.21 ± 0.05) × 104 M−1 with detection limit 92.7 nM. On addition of S2− to the FO511–Hg2+complex, the fluorescence intensity was totally quenched due to removal of Hg2+ from the complex by S2− ion arising out of stronger affinity of Hg2+ towards S2− resulting concomitant formation of ring closed non-fluorescent form, FO511. The tentative coordination environment in the FO511–Hg2+complex was established by DFT studies. FO511 exhibits low cytotoxicity and cell permeability, which makes it capable for bioimaging applications in living HepG2 cells.

Chemo- and Site-Selective Lysine Modification of Peptides and Proteins under Native Conditions Using the Water-Soluble Zolinium

Site-selective lysine modification of peptides and proteins in aqueous solutions or in living cells is still a big challenge today. Here, we report a novel strategy to selectively quinolylate lysine residues of peptides and proteins under native conditions without any catalysts using our newly developed water-soluble zoliniums. The zoliniums could site-selectively quinolylate K350 of bovine serum albumin and inactivate SARS-CoV-2 3CLpro via covalently modifying two highly conserved lysine residues (K5 and K61). In living HepG2 cells, it was demonstrated that the simple zoliniums (5b and 5B) could quinolylate protein lysine residues mainly in the nucleus, cytosol, and cytoplasm, while the zolinium-fluorophore hybrid (8) showed specific lysosome-imaging ability. The specific chemoselectivity of the zoliniums for lysine was validated by a mixture of eight different amino acids, different peptides bearing potential reactive residues, and quantum chemistry calculations. This study offers a new way to design and develop lysine-targeted covalent ligands for specific application.

Implication toward a simple strategy to generate pH tunable FRET-based biosensing

The present contribution depicts a unique approach to generate tunable Förster resonance energy transfer (FRET) emission with variation of pH of the medium. The pH sensitive absorption of Doxorubicin leads to modification of spectral overlap between emission spectra of donor (Pyrazoline) and absorption spectra of acceptor (Doxorubicin) thereby sensing maximum FRET efficiency in an optimum pH (near pKa of Doxorubicin). This drug molecule exhibits an instantaneous conformation change at a particular pH, which consequences on abrupt ON-and-OFF FRET efficiency. At elevated pH, both the drug molecules exhibit conformational change and form stable fluorescent exciplex, switching off the FRET emission. Confocal fluorescence images of live HepG2 cells imply that the sensor can proficiently go through the cell membrane and can be applied in the controlled delivery of drug to the tumor cell lines.