Living HeLa Cells Research Articles

Two reasonably designed polarity-viscosity sensitive fluorescent probes with large Stokes shift for lighting up lipid droplets in cells

Lipid droplets (LDs) are involved in a variety of complex life activities in cells, which are closely related to human diseases including obesity, diabetes and cancer. LDs are generally considered as a kind of organelle with extremely low water content and high viscosity. Herein, two novel probes (E)-2-(3-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (LDP-1) and (E)-2-(3-(4-(diethylamino)styryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (LDP-2) were synthesized, which exhibited large Stokes shifts of 4758 cm−1 and 3986 cm−1 respectively. The two probes emitted strongly in hydrophobic and high viscosity environments. Moreover, bio-probes LDP-1 and LDP-2 exhibited low biological toxicity, and good selectivity. The two probes were also fit for selectively monitoring LDs distribution in living HeLa cells.

Coumarin 1,4-enedione for selective detection of hydrazine in aqueous solution and fluorescence imaging in living cells.

Hydrazine is a widely used but highly toxic chemical reagent, and the development of a fluorescent probe for hydrazine detection is very meaningful. In this study, a novel coumarin-derived fluorescent probe containing a 1,4-enedione moiety for hydrazine detection was developed. The recognition of hydrazine with the probe brings about obvious fluorescence enhancement over other environmentally relevant ions and amine-containing species. The limit of detection for hydrazine is 2.7×10-8 M in aqueous solution. The fluorescence enhancement was ascribed to the cyclization reaction of the 1,4-enedione moiety of the probe and hydrazine which form a six-membered pyridazine ring and intramolecular charge transfer (ICT) mechanism. The mass spectrometry (MS), nuclear magnetic resonance (NMR) analysis and theoretical calculations confirmed the recognition produced. The probe can be used to determine trace hydrazine in real water samples. More importantly, the probe also showed good potential in detecting hydrazine by imaging of living HeLa cells.

Near-infrared fluorescence probe with a large Stokes shift for selectively imaging of hydrogen peroxide in living cells and in vivo

In this work, we have designed and synthesized a large stokes shift and near-infrared (NIR) probe DCM-HNU for visualization of H2O2 in both living HeLa cells and zebra fish. The probe DCM-HNU was structured based on phenylborate pincinol ester as the recognition group connecting DCM-OH via nucleophilic substitution reaction. The probe possessed large stokes shift (205 nm), near-infrared (NIR) emission (658 nm). 0.17 μM detection Limit, 10,550 L·mol−1cm−1 molar extinction coefficient, 2.06 × 10−3 S−1 reaction rate constant, and excellent selectivity proved the condition for practical application. There was a good linear relationship between fluorescence intensity and H2O2 concentration from 0 to 200 μM (R2 = 0.9955) to facilitate quantitative analysis of H2O2. More importantly, the probe is able to visualize exogenous and endogenous H2O2 in living HeLa cells and zebra fish. The superior properties of the probe provide potential for exploring the function and participation of H2O2 in biological systems.

Facile solvothermal synthesis of ultrathin spinel ZnMn2O4 nanospheres: An efficient electrocatalyst for in vivo and in vitro real time monitoring of H2O2

• The ultrathin ZnMn 2 O 4 (ZMO) nanospheres were prepared by facile solvothermal method. • The ZMO exhibited wide linearity (20 nM-10 mM) with low detection limit (8.3 nM). • The developed biosensor showed rapid response (> 3s), high selectivity and stability. • The proposed sensor was applied for in-vivo and in-vitro real-time analysis of H 2 O 2. . Hydrogen peroxide (H 2 O 2 ) is a renowned industrial oxidant that plays a significant role in cell biology, and pathogenesis. On this note, we propose a simple Zinc Manganese Oxide (ZnMn 2 O 4 ; ZMO) based H 2 O 2 sensor for rapid in-situ tracking and determination of H 2 O 2 from live cells, and bio-samples. The ZMO catalyst have been synthesized via simple scalable protocol; surface morphology, elemental composition, electronic states, crystallinity, and other physiochemical properties are studied and discussed in-detail using various spectroscopic tools. The ZMO modified electrode displays an excellent electrochemical activity toward H 2 O 2 sensing with a wide dynamic range (20 nM-10 mM), low detection limit (8.3 nM), excellent selectivity, reproducibility, operational stability, and rapid response time (<3 s). Further, the ZMO modified electrode have been found successful at in-vivo real-time tracking of H 2 O 2 released from live HeLa cells, and in-vitro analysis of H 2 O 2 spiked in water, tooth paste, and human serum samples.

Two simple but effective turn-on benzothiazole-based fluorescent probes for detecting hydrogen sulfide in real water samples and HeLa cells

Two simple turn-on fluorescent probes, containing a benzothiazole and the 2,4-dinitrobenzenesulfonyl group, were designed for detecting H2S. Two probes exhibited good selectivity and high sensitivity, which were applied to detect the H2S in real water samples. Probe P2 with a positive charge had better solubility than probe P1 in water; therefore, probe P2 was successfully applied to detect both the endogenous and exogenous H2S in lysosomes of living HeLa cells.

Bioorthogonal Photocatalytic Decaging-Enabled Mitochondrial Proteomics.

Spatiotemporally resolved dissection of subcellular proteome is crucial to our understanding of cellular functions in health and disease. We herein report a bioorthogonal and photocatalytic decaging-enabled proximity labeling strategy (CAT-Prox) for spatiotemporally resolved mitochondrial proteome profiling in living cells. Our systematic survey of the photocatalysts has led to the identification of Ir(ppy)2bpy as a bioorthogonal and mitochondria-targeting catalyst that allowed photocontrolled, rapid rescue of azidobenzyl-caged quinone methide as a highly reactive Michael acceptor for proximity-based protein labeling in mitochondria of live cells. Upon careful validation through in vitro labeling, mitochondria-targeting specificity, in situ catalytic activity as well as protein tagging, we applied CAT-Prox for mitochondria proteome profiling in living Hela cells as well as hard-to-transfect macrophage RAW264.7 cells with approximately 70% mitochondria specificity observed from up to 300 proteins enriched. Finally, CAT-Prox was further applied to the dynamic dissection of mitochondria proteome of macrophage cells upon lipopolysaccharide stimulation. By integrating photocatalytic decaging chemistry with proximity-based protein labeling, CAT-Prox offers a general, catalytic, and nongenetic alternative to the enzyme-based proximity labeling strategies for diverse live cell settings.

A novel and stable fluorescent probe for tracking Hg2+ with large Stokes shift and its application in cell imaging

Giving the fact that mercury ions (Hg2+) is highly toxic, migratory and bioaccumulative and even very small amounts of mercury can cause serious damage to health, resulting in many diseases, such as abdominal pain, renal failure, nervous system damage. The content of mercury in drinking water quality standard of our country has been strictly limited. Therefore, it is of good research interest to develop a stable fluorescent probe capable of detecting the presence of mercury in biological cells. In this study, a novel fluorescent probe based on isophoronitriles scaffold, DNC-Hg, was designed and synthesized for monitoring mercury ion in living HeLa cells. The good properties of the probe may be attributed to the unique strong electron-absorbing group in the structural design, the good conjugation effect, and the mature Hg2+ recognition site. The probe exhibited good selectivity and stability, large Stokes shift(174 nm) and low cytotoxicity. Furthermore, this stable probe DNC-Hg could be used for cellular imaging.

A novel [1,2,4]triazolo[1,5-a]pyrimidine derivative as a fluorescence probe for specific detection of Fe3+ ions and application in cell imaging

The detection of metal ions is of particular importance for monitoring environmental pollution and life metabolic activities. However, it is still a challenge to achieve Fe3+ detection with specific sensitivity and rapid response, especially in the presence of chelating agents for Fe3+ ions. Herein, a novel fluorescence probe for Fe3+, i.e., amide derivative of [1,2,4]triazolo[1,5-a] pyrimidine (TP, Id), was synthesized, featuring specific Fe3+ selectivity, rapid quenching (5 s), low limit of detection (0.82 μM), good permeability and low cytotoxicity. More importantly, Id can be used to identify and detect Fe3+ in the presence of existing strong chelating agents (e.g., EDTA) for Fe3+ ions. The results show that the as-synthesized fluorescence probe is particularly suitable as a bioimaging reagent to monitor intracellular Fe3+ in living HeLa cells. Furthermore, we proposed the binding mode for Id with Fe3+ ions and the light-emitting mechanism through high-resolution mass spectra and density function theory calculations, respectively. An Id-based test paper can be used to rapidly identify Fe3+. These results are expected to improve the development of new sensitive and specific fluorescent sensors for Fe3+.

A new recognition moiety diphenylborinate in the detection of pyruvate via Lewis acid/base sensing pathway and its bioimaging applications

Developing new reaction based recognizing units can enhance the specificity of target analyte molecules in practical applications of real samples and biosystems. Therefore, introducing a recognizing moiety diphenylborinate was encountered for the detection of pyruvate biomolecule through Lewis acid-base reaction based sensing strategy. The construction of the Schiff-base back bone between quinoline and N-(diethylamino)salicylaldehyde-diphenylborinate (QSB) were expressed the red shift from blue emission of quinoline in to green as that of dative bond developed between Schiff base nitrogen and boron atoms. The new sensing approach was involved such a way that fluorophore QSB is a Lewis acid while pyruvate acts as Lewis base, where the elimination of Lewis pair produced a weak green fluorescence including the formation of quinoline, N-(diethylamino)salicylaldehyde (QS). The switching products were witnessed through 1H NMR titration, HR-MS and FT-IR studies. The good selectivity and interference ability were achieved in presence of 1000-fold excess of possible interferences with LOD of 16 nM. Moreover, the tracking ability of the probe was employed towards pyruvate in live HeLa cell imaging for evaluating an exogenous and endogenous signals producing ability and its mitochondria targeting property was investigated successfully. Further, the practical utility of the probe was tested with milk samples and obtained good recovery results.

A turn-on fluorescent probe via substitution-rearrangement for highly sensitive and discriminative detection of cysteine and its imaging in living cells

Biothiols play an important role in many physiological and pathological processes, especially in the occurrence of oxidative stress caused by abnormal cysteine (Cys) concentration. Therefore, it is particularly critical to develop a method that can specifically identify Cys to avoid interference from other biological analytes. However, most Cys-specific fluorescent probes are difficult to distinguish between homocysteine (Hcy) and glutathione (GSH). In this work, to avoid the interference of Hcy and GSH, we developed a fluorescent probe triarylimidazole-naphthalimide-piperazine-sulfonyl benzoxadiazole (TNP-SBD-Cl) based on fluorescence resonance energy transfer (FRET) on platform of naphthalimide-sulfonyl benzoxadiazole (SBD), the main SBD 4-chlorine groups have mild reactivity to undergo substitution and rearrangement to distinguish Hcy and GSH. The TNP-SBD-Cl response to Cys would turn on FRET and generate a new yellow fluorescence with a large Stokes shift (157 nm), and with excellent selectivity and low detection limit (0.87 μM). Moreover, TNP-SBD-Cl can be used to monitor Cys in living HeLa cells with low cytotoxicity, suggesting that it has markedly diagnostic significance in physiological and pathological processes.

Optical section structured illumination-based Förster resonance energy transfer imaging.

Förster resonance energy transfer (FRET) microscopy is an important tool suitable for studying molecular interactions in living cells. Optical section structured illumination microscopy (OS-SIM), like confocal microscopy, has about 200 nm spatial resolution. In this report, we performed quantitative 3-cube FRET imaging in OS-SIM mode and widefield microscopy (WF) mode, respectively, for living cells expressing FRET constructs consisting of Cerulean (C, donor) and Venus (V, acceptor). OS-SIM images exhibited higher resolution than WF images. Four spectral crosstalk coefficients measured under OS-SIM mode are consistent with those measured under WF mode. Similarly, the system calibration factors G and k measured under OS-SIM mode were consistent with those measured under WF mode. The measured FRET efficiency (E) values of C32V and C17V as well as C5V constructs, standard FRET plasmids, in living Hela cells were , and , and the measured acceptor-to-donor concentration ratios ( ) were , , and , consistent with the reported values. Collectively, our data demonstrates that OS-SIM can be integrated into FRET microscopy to build an OS-SIM-FRET with confocal microscopy-like resolution.

A Novel Fluorescence Probe for the Reversible Detection of Bisulfite and Hydrogen Peroxide Pair in Vitro and in Vivo.

The detection of changes in the reactive oxygen species (ROS)/reactive sulfur species (RSS) couple is important for studying the cellular redox state. Herein, we developed a 1,8-naphthalimide-based fluorescence probe (NI) for the reversible detection of bisulfite (HSO3 - ) and hydrogen peroxide (H2 O2 ) in vitro and in vivo. NI has been designed with a reactive ethylene unit which specifically reacts with HSO3 - by a Michael addition reaction mechanism, resulting in the quenching of yellow fluorescence at 580 nm and the appearing of green fluorescence at 510 nm upon excitation at 500 nm and 430 nm, respectively. The addition product (NI-HSO3 ) could be specifically oxidized to form the original C=C bond of NI, recovering the fluorescence emission and color. The detection limits of NI for HSO3 - and NI-HSO3 for H2 O2 were calculated to be 2.05 μM and 4.23 μM, respectively. The reversible fluorescence response of NI towards HSO3 - /H2 O2 couple can be repeated for at least five times. NI is reliable at a broad pH range (pH 3.0-11.5) and features outstanding selectivity, which enabled its practical applications in biological and food samples. Monitoring the reversible and dynamic inter-conversion between HSO3 - and H2 O2 in vitro and in vivo has been verified by fluorescence imaging in live HeLa cells, adult zebrafish and nude mice. Moreover, NI has been successfully applied to detect of HSO3 - levels in food samples.

Rapid ensemble measurement of protein diffusion and probe blinking dynamics in cells

We present a fluorescence fluctuation image correlation analysis method that can rapidly and simultaneously measure the diffusion coefficient, photoblinking rates, and fraction of diffusing particles of fluorescent molecules in cells. Unlike other image correlation techniques, we demonstrated that our method could be applied irrespective of a nonuniformly distributed, immobile blinking fluorophore population. This allows us to measure blinking and transport dynamics in complex cell morphologies, a benefit for a range of super-resolution fluorescence imaging approaches that rely on probe emission blinking. Furthermore, we showed that our technique could be applied without directly accounting for photobleaching. We successfully employed our technique on several simulations with realistic EMCCD noise and photobleaching models, as well as on Dronpa-C12-labeled β-actin in living NIH/3T3 and HeLa cells. We found that the diffusion coefficients measured using our method were consistent with previous literature values. We further found that photoblinking rates measured in the live HeLa cells varied as expected with changing excitation power.

An AIE-active probe for selective fluorometric–colorimetric detection of HSO3− in aqueous solution and real samples

We report herein a new biphenylcarbonitrile derivative, 4′-hydroxy-3′ -((2-imidazolylimino) methyl)-4-biphenyl carbonitrile (HIBC), which interestingly exhibits an aggregation-induced emission property. HIBC was employed for selective dual mode (fluorimetric and colorimetric) assay of HSO3− ions in nearly 100% aqueous medium at pH 7.4. The nucleophilic addition of HSO3− on the imino group in HIBC breaks its π-electron conjugation and results in a noticeable absorption and fluorescence spectral changes accompanied by a visible color change. Moreover, the formation of anionic addition product weakened the aggregation effect and meanwhile inhibited ESIPT process in HIBC, causing quenching of keto fluorescence of the probe. The probe shows good selectivity for HSO3− ions over all other competitive anions, which renders it a promising candidate for ratiometric fluorescent assay of HSO3− in aqueous systems. The limit of detection is 1.83 × 10−7 mol/L. For the practical applicability, handy paper strips were fabricated from HIBC for convenient recognition of HSO3−. Additionally, HIBC was applied for the living HeLa cells bio-imaging.

Two novel “turn on” fluorescent probes for monitoring hypochlorite in living HeLa cells

In this study, two benzothiazole derivative fluorescent probes including 2-(4-(7-(4-(diphenylamino)phenyl)benzo[c][1,2,5]thiadiazol-4-yl)benzylidene)malononitrile (abbreviated as TBDP) and 2-((5-(7-(4-(diphenylamino)phenyl)benzo- [c][1,2,5]thiadiazol-4-yl)thiophen-2-yl)methylene)malononitrile (TBDT) for ClO- were designed and synthesized. TBDP and TBDT exhibited red-light emission in THF solution. The emission band was centered at 605 nm for TBDP and 620 nm for TBDT with fluorescent quantum yield (Φ) being 4.82% and 4.66%, respectively. They can be used as highly sensitive ‘‘turn on’’ probes for hypochlorite (ClO-) with a fast response time and high selectivity. Upon the addition of ClO-, the Φ values of TBDP and TBDT were lightened to 48.76% and 34.77%. The limit of detection (LOD) value was captured as low as 75 nM and 33 nM for TBDP and TBDT, respectively. The recognition mechanism of the probes was researched through 1H NMR, HRMS and theoretical calculation. More importantly, TBDT can effectively visualize trace amount of exogenous ClO- in living HeLa cells and identify endogenous ClO- in living Raw 264.7 cells. Overall, the probes described herein will efficiently optimize molecular design strategy to supply low LOD for ClO-.

Near-infrared emissive polymer-coated IR-820 nanoparticles assisted photothermal therapy for cervical cancer cells.

Photothermal therapy (PTT) has attracted wide attention due to its noninvasiveness and its thermal ablation ability. As photothermal agents are crucial factor in PTT, those with the characteristics of biocompatibility, non-toxicity and high photothermal stability have attracted great interest. In this work, new indocyanine green (IR-820) was utilized as a photothermal agent and near-infrared (NIR) fluorescence imaging nanoprobe. To improve the biocompatibility, poly(styrene-co-maleic anhydride) (PSMA) was utilized to encapsulate the IR-820 molecules to form novel IR-820@PSMA nanoparticles (NPs). Then, the optical and thermal properties of IR-820@PSMA NPs were studied in detail. The IR-820@PSMA NPs showed excellent photothermal stability and biocompatibility. The cellular uptaking ability of the IR-820@PSMA NPs was further confirmed in HeLa cells by the NIR fluorescent confocal microscopic imaging technique. The IR-820@PSMA NPs assisted PTT of living HeLa cells was conducted under 793 nm laser excitation, and a high PTT efficiency of 73.3% was obtained.

The impact of TiO2 nanoparticle exposure on transmembrane cholesterol transport and enhanced bacterial infectivity in HeLa cells

We have previously shown that exposure to TiO2 nanoparticles (NPs) reduces the resistance of HeLa cells to bacterial infection. Here we demonstrate that the increased infectivity is associated with enhanced asymmetry in the cholesterol distribution. We applied a live cell imaging method which uses tunable orthogonal cholesterol sensors to visualize and quantify in-situ cholesterol distribution between the two leaflets of the plasma membrane (PM). In the control culture, we found marked transbilayer asymmetry of cholesterol, with the concentration in the outer plasma membrane (OPM) being 13 ± 2-fold higher than that in the inner plasma membrane (IPM). Exposure of the culture to 0.1 mg/mL of rutile TiO2 NPs increased the asymmetry such that the concentration in the OPM was 51 ± 10 times higher, while the total cholesterol content increased only 21 ± 2%. This change in cholesterol gradient may explain the increase in bacterial infectivity in HeLa cells exposed to TiO2 NPs since many pathogens, including Staphylococcus aureus used in the present study, require cholesterol for proper membrane attachment and virulence. RT-PCR indicated that exposure to TiO2 was responsible for upregulation of the ABCA1 and ABCG1 mRNAs, which are responsible for the production of the cholesterol transporter proteins that facilitate cholesterol transport across cellular membranes. This was confirmed by the observation of an overall decrease in bacterial infection in ABCA1 knockout or methyl-β-cyclodextrin-treated HeLa cells, as regardless of TiO2 NP exposure. Hence rather than preventing bacterial infection, TiO2 nanoparticles upregulate genes associated with membrane cholesterol production and distribution, hence increasing infectivity. Statement of significanceA great deal of work has been done regarding the toxicology of the particles, especially focusing on detrimental outcomes associated with reactive oxygen species (ROS) production. In this paper we show unambiguously a very surprising result, namely the ability of these particles to enhance bacterial infection even at very small exposure levels, where none of the deleterious effects of ROS products can yet be detected. Using a new imaging technique, we are able to demonstrate, in operando, the effect of the particles on cholesterol generation and distribution in live HeLa cells. This paper also represents the first in a series where we explore other consequences of increased membrane cholesterol, due to particle exposure, which are known to have multiple other consequences on human tissue function and development.

A highly-sensitive genetically encoded temperature indicator exploiting a temperature-responsive elastin-like polypeptide

Genetically encoded temperature indicators (GETIs) allow for real-time measurement of subcellular temperature dynamics in live cells. However, GETIs have suffered from poor temperature sensitivity, which may not be sufficient to resolve small heat production from a biological process. Here, we develop a highly-sensitive GETI, denoted as ELP-TEMP, comprised of a temperature-responsive elastin-like polypeptide (ELP) fused with a cyan fluorescent protein (FP), mTurquoise2 (mT), and a yellow FP, mVenus (mV), as the donor and acceptor, respectively, of Förster resonance energy transfer (FRET). At elevated temperatures, the ELP moiety in ELP-TEMP undergoes a phase transition leading to an increase in the FRET efficiency. In HeLa cells, ELP-TEMP responded to the temperature from 33 to 40 °C with a maximum temperature sensitivity of 45.1 ± 8.1%/°C, which was the highest ever temperature sensitivity among hitherto-developed fluorescent nanothermometers. Although ELP-TEMP showed sensitivity not only to temperature but also to macromolecular crowding and self-concentration, we were able to correct the output of ELP-TEMP to achieve accurate temperature measurements at a subcellular resolution. We successfully applied ELP-TEMP to accurately measure temperature changes in cells induced by a local heat spot, even if the temperature difference was as small as < 1 °C, and to visualize heat production from stimulated Ca2+ influx in live HeLa cells induced by a chemical stimulation. Furthermore, we investigated temperatures in the nucleus and cytoplasm of live HeLa cells and found that their temperatures were almost the same within the temperature resolution of our measurement. Our study would contribute to better understanding of cellular temperature dynamics, and ELP-TEMP would be a useful GETI for the investigation of cell thermobiology.

Development of a ratiometric fluorescent probe with large Stokes shift and emission wavelength shift for real-time tracking of hydrazine and its multiple applications in environmental analysis and biological imaging

As a tremendously noxious and extensively utilized chemical reagent, hydrazine (N2H4) has become a serious threat to ecosystem and human health. Thus, it is desirable to exploit an efficient method for real-time tracking of hydrazine. Here, a novel ratiometric fluorescent probe PBQ-AB for hydrazine was rationally constructed from isolongifolanone. This probe displayed an extremely large Stokes shift of 230 nm and could selectively recognize hydrazine in the presence of other competitive species within an extremely short time ( 40 s). PBQ-AB also displayed some fascinating merits in the detection of hydrazine, including low detection limit (48 nM), wide pH range (5–12), excellent photostability (>240 min), and well-resolved emission wavelength shift (148 nm). Moreover, this probe was utilized to fabricate a ready-to-use electrospinning nanofibrous membrane for convenient detection of hydrazine vapor by virtue of smartphone. Furthermore, PBQ-AB was capable of determining hydrazine contaminant in environmental soil and water samples. Additionally, its favorable performance for detecting hydrazine was successfully demonstrated in live HeLa cells as well as in live Arabidopsis thaliana tissues, manifesting its promising application for labeling hydrazine in living systems. Therefore, we believed that this probe has great potential in environmental analysis and health supervision.

A isophorone based colorimetric and ratiometric probe: Synthesis and rapid detection of cyanide in aqueous medium and its application in bioimaging

A simple colorimetric and fluorescent sensor for cyanide based on phenolic substituted dicyanoisophorone derivative (2) has been synthesized. The absorption spectra of chemosensor 2 showed large bathochromic shift (ca. 240 nm) upon titration with CN− while the solution color changed from yellow to green. The absorption ratios (A685/A446) responses of chemosensor 2 showed a favorable linear relationship with the CN− concentration and the detection limitation had been calculated to be 0.15 μM. Meanwhile, the fluorescence intensity of 2 can be quenched upon reacting with CN− over other anions. More importantly, chemosensor 2 can react instantaneously with cyanide in aqueous solution, and to visualize CN− in living HeLa cells by means of dramatic decrease of fluorescence intensity. Thus, the present sensor could be applicable in detecting cyanide ion both in natural water samples and biological systems conveniently.