Novel fluorescence detection technique for non-contact temperature sensing in microchip PCR

DNA amplification using Polymerase Chain Reaction (PCR) in a small volume is used in Lab-on-a-chip systems involving DNA manipulation. For few microliters of volume of liquid, it becomes difficult to measure and monitor the thermal profile accurately and reproducibly, which is an essential requirement for successful amplification. Conventional temperature sensors are either not biocompatible or too large and hence positioned away from the liquid leading to calibration errors. In this work we present a fluorescence based detection technique that is completely biocompatible and measures directly the liquid temperature. PCR is demonstrated in a 3 ILL silicon-glass microfabricated device using non-contact induction heating whose temperature is controlled using fluorescence feedback from SYBR green I dye molecules intercalated within sensor DNA. The performance is compared with temperature feedback using a thermocouple sensor. Melting curve followed by gel electrophoresis is used to confirm product specificity after the PCR cycles. (c) 2007 Elsevier B.V. All rights reserved.

A cationic cholesterol based nanocarrier for the delivery of p53-EGFP-C3 plasmid to cancer cells

The p53 protein mediated anti-tumor strategy is limited due to the lack of suitable delivery agent with insignificant immunogenic response, serum compatibility, and early and easy detection of the transfected cell population. To overcome these problems, we generated a p53-EGFP-C3 fusion construct which expressed easily detectable green fluorescence protein (GFP) and allowed an estimation of p53 mediated anti-tumor activity. A mixture of cationic cholesterol gemini (Choi-5L) with natural lipid, DOPE (molar ratio 1:4), acronymed as Chol-5LD, formed a nano-liposome as characterized by various physical methods. The prepared clone was evaluated for the expression of GFP and functional p53 in HeLa and two additional cell lines with varied p53 status namely, H1299 (p53(-/-)) and HEK293T (p53(+/+)). Transfected cells were screened using RT-PCR, Western blotting, FACS analysis, MTT, Trypan blue assay and visualized under a fluorescence microscope. The p53-EGFP-C3 fusion protein induced apoptosis in cancer cells as evident from DNA fragmentation, cell cycle analysis, Annexin-V staining and PARP cleavage assays. The transfection and apoptosis induction efficiency of Chol-5LD was significantly higher than commercial reagents Lipofectamine2000 and Effectene irrespective of the cell lines examined. Further it significantly decreases the xenograft tumor volume in nude mice tumors via apoptosis as observed in H&E staining. (C) 2013 Elsevier Ltd. All rights reserved.

DNA binding properties of some cationic acridine derivatives

Four cationic acridine derivatives have been synthesized. The positively charged amine residue in one of these is connected directly on to the acridine nucleus and in three other acridines, the amines are connected via a 9-CH2 unit to acridine. We have investigated the binding of these acridines with mammalian DNA by absorption titration, UV- and induced-CD spectroscopy and competitive ethidium bromide displacement fluorescence assay. The effects on the DNA duplex denaturation melting temperatures upon binding of each one of these are also examined. The results obtained herein clearly show that the introduction of a -CH2 group in the im mediate vicinity of the interrelation moiety introduces alterations in the DNA binding characteristics of the resulting acridines.

Syntheses, Transfection Efficacy and Cell Toxicity Properties of Novel Cholesterol-based Gemini Lipids having Hydroxyethyl Head group

We have synthesized five new cholesterol based gemini cationic lipids possessing hydroxyethyl (-CH2CH2OH) function on each head group, which differ in the length of the polymethylene spacer chain. These gemini lipids are important for gene delivery processes as they possess pre-optimized molecular features, e. g., cholesterol backbone, ether linkage and a variable spacer chain between both the headgroups of the gemini lipids. Cationic liposomes were prepared from each of these lipids individually and as a mixture of individual cationic gemini lipid and 1,2-dioleoyl phosphatidylethanolamine (DOPE). Each gemini lipid based formulation induced better transfection activity than that of their monomeric counterpart. One such gemini lipid with a -(CH2)(12)-spacer, HG-12, showed dramatic increase in the mean fluorescence intensity due to the expression of green-fluorescence protein (GFP) in the presence of 10% FBS compared to the conditions where there was no serum. Other gemini lipids retained their gene transfection efficiency without any marked decrease in the presence of serum. The only exception was seen with the gemini with a -(CH2)(3)-spacer, HG-3, which on gene transfection in the presence of 10% FBS lost similar to 70% of its transfection efficiency. Overall the gemini lipid with a -(CH2)(5)-spacer, HG-5, showed the highest transfection activity at N/P (lipid/DNA) ratio of 0.5 and lipid : DOPE molar ratio of 2. Upon comparison of the relevant parameters, e. g., %-transfected cells, the amount of DNA transfected to each cell and %-cell viability all together against Lipofectamine 2000, one of the best commercial transfecting agents, the optimized lipid formulation based on DOPE/HG-5 was found to be comparable. In terms of its ability to induce gene-transfer in the presence of serum and shelf-life DOPE/HG-5 liposome was found to be superior to its commercial counterpart. Confocal imaging analysis confirmed that in the presence of 10% serum using a Lipid : DOPE of 1 : 4 and N/P charge ratio of 0.75 with 1.2 mu g DNA per well, HG-5 is better than Lipofectamine 2000.

Efficacious redox-responsive gene delivery in serum by ferrocenylated monomeric and dimeric cationic cholesterols

Herein, we present the design and synthesis of new redox-active monomeric and dimeric (gemini) cationic lipids based on ferrocenylated cholesterol derivatives for gene delivery. The cationic cholesterols are shown to be transfection efficient after being formulated with the neutral helper lipid DOPE in the presence of serum (FBS). The redox activity of the resulting co-liposomes and their lipoplexes could be regulated using the alkanyl ferrocene moiety attached to the ammonium head groups of the cationic cholesterols. Atomic force microscopy (AFM), dynamic light scattering (DLS) and zeta potential measurements were performed to characterize the co-liposomal aggregates and their complexes with pDNA. The transfection efficiency of lipoplexes could be tuned by changing the oxidation state of the ferrocene moiety. The gene transfection capability was assayed in terms of green fluorescence protein (GFP) expression using pEGFP-C3 plasmid DNA in three cell lines of different origins, namely Caco-2, HEK293T and HeLa, in the presence of serum. The vesicles possessing ferrocene in the reduced state induced an efficient transfection, even better than a commercial reagent Lipofectamine 2000 (Lipo 2000) as evidenced by flow cytometry and fluorescence microscopy. All the co-liposomes containing the oxidized ferrocene displayed diminished levels of gene expression. Gene transfection events from the oxidized co-liposomes were further potentiated by introducing ascorbic acid (AA) as a reducing agent during lipoplex incubation with cells, leading to the resumption of transfection activity. Assessment of transfection capability of both reduced and oxidized co-liposomes was also undertaken following cellular internalization of labelled pDNA using confocal microscopy and flow cytometry. Overall, we demonstrate here controlled gene transfection activities using redox-driven, transfection efficient cationic monomeric and dimeric cholesterol lipids. Such systems could be used in gene delivery applications where transfection needs to be performed spatially or temporally.

Substituent effect on fluorescence signaling of the cell permeable HSO4- receptors through single point to ratiometric response in green solvent

Two new 2-(2-aminophenyl)benzimidazole-based HSO4- ion selective receptors, 6-(4-nitrophenyl)-5,6-dihydrobenzo4,5]imidazo1,2-c]quinazoline (L1H) and 6-(4-methoxyphenyl)-5,6-dihydrobenzo4,5]imidazo1,2-c] quinazoline (L2H), and their 1 : 1 molecular complexes with HSO4- were prepared in a facile synthetic method and characterized by physicochemical and spectroscopic techniques along with the detailed structural analysis of L1H by single crystal X-ray crystallography. Both receptors (L1H and L2H) behave as highly selective chemosensor for HSO4- ions at biological pH in ethanol-water HEPES buffer (1/5) (v/v) medium over other anions such as F-, Cl-, Br-, I-, AcO-, H2PO4-, N-3(-) and ClO4-. Theoretical and experimental studies showed that the emission efficiency of the receptors (L1H and L2H) was tuned successfully through single point to ratiometric detection by employing the substituent effects. Using 3 sigma method the LOD for HSO4- ions were found to be 18.08 nM and 14.11 nM for L1H and L2H, respectively, within a very short responsive time (15-20 s) in 100 mM HEPES buffer (ethanol-water: 1/5, v/v). Comparison of the utility of the probes (L1H and L2H) as biomarkers for the detection of intracellular HSO4- ions concentrations under a fluorescence microscope has also been included and both probes showed no cytotoxic effect.

Differential fluorescence of the chromocenters and nucleolar equivalents of the yeast nucleus in acridine orange

The nucleus with its limiting membrane and organelles was visible in the majority of the yeast cells stained vitally with the fluorochrome, acridine orange, at a dilution of 1 in 40,000. The intra-nuclear structures could be distinguished by their differential fluorescence. The chromocenters were green while the nucleolar equivalents were orange. The vacuole showed no fluorescence.

Analytical solutions for diffuse fluorescence spectroscopy/imaging in biological tissues. Part I: zero and extrapolated boundary conditions

The mathematical model for diffuse fluorescence spectroscopy/imaging is represented by coupled partial differential equations (PDEs), which describe the excitation and emission light propagation in soft biological tissues. The generic closed-form solutions for these coupled PDEs are derived in this work for the case of regular geometries using the Green's function approach using both zero and extrapolated boundary conditions. The specific solutions along with the typical data types, such as integrated intensity and the mean time of flight, for various regular geometries were also derived for both time-and frequency-domain cases. (C) 2013 Optical Society of America

Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy

Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4 pi geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as compared to 690 nm of the state-of-art confocal system. Reconstruction of experimentally obtained 2PE - 4pi data of green fluorescent protein (GFP)-tagged mitocondrial network shows near elimination of artifacts arising out of side-lobes. Proposed technique may find interesting application in fluorescence microscopy, nano-lithography, and cell biology. (C) 2013 AIP Publishing LLC.

In Vivo Genotoxicity Assessment of Titanium Dioxide Nanoparticles by Allium cepa Root Tip Assay at High Exposure Concentrations

The industrial production and commercial applications of titanium dioxide nanoparticles have increased considerably in recent times, which has increased the probability of environmental contamination with these agents and their adverse effects on living systems. This study was designed to assess the genotoxicity potential of TiO2 NPs at high exposure concentrations, its bio-uptake, and the oxidative stress it generated, a recognised cause of genotoxicity. Allium cepa root tips were treated with TiO2 NP dispersions at four different concentrations (12.5, 25, 50, 100 mu g/mL). A dose dependant decrease in the mitotic index (69 to 21) and an increase in the number of distinctive chromosomal aberrations were observed. Optical, fluorescence and confocal laser scanning microscopy revealed chromosomal aberrations, including chromosomal breaks and sticky, multipolar, and laggard chromosomes, and micronucleus formation. The chromosomal aberrations and DNA damage were also validated by the comet assay. The bio-uptake of TiO2 in particulate form was the key cause of reactive oxygen species generation, which in turn was probably the cause of the DNA aberrations and genotoxicity observed in this study.

Photocytotoxic oxovanadium(IV) complexes of ferrocenyl-terpyridine and acetylacetonate derivatives

Oxovanadium(IV) complexes VO(Fc-tpy)(acac)](ClO4) (1), VO(Fc-tpy)(nap-acac)](ClO4) (2), VO(Fc-tpy)(py-acac)](ClO4) (3) and VO(Ph-tpy)(py-acac)](ClO4) (4) of 4'-ferroceny1-2,2':6',2 `'-terpyridine (Fc-tpy) and 4'-phenyl-2,2':6',2 `'-terpyridine (Ph-tpy) having monoanionic acetylacetonate (acac), naphthylacetylacetonate (nap-acac) or pyrenylacetylacetonate (py-acac) ligand were prepared, characterized and their photocytotoxicity in visible light studied. The ferrocenyl complexes 1-3 showed an intense charge transfer band near 585 nm in DMF and displayed Fc(+)/Fc and V(IV)/V(III) redox couples near 0.66 V and -0.95 V vs. SCE in DMF-0.1 M TBAP. The complexes as avid binders to calf thymus DNA showed significant photocleavage of plasmid DNA in green light (568 nm) forming center dot OH radicals. The complexes that are photocytotoxic in HeLa and MCF-7 cancer cells in visible light (400-700 nm) with low dark toxicity remain nontoxic in normal fibroblast 3T3 cells. ICP-MS and fluorescence microscopic studies show significant cellular uptake of the complexes. Photo-irradiation of the complexes causes apoptotic cell death by ROS as evidenced from the DCFDA assay. (C) 2015 Elsevier Masson SAS. All rights reserved.

Stimuli-responsive colorimetric and NIR fluorescence combination probe for selective reporting of cellular hydrogen peroxide

Hydrogen peroxide (H2O2) is a key reactive oxygen species and a messenger in cellular signal transduction apart from playing a vital role in many biological processes in living organisms. In this article, we present phenyl boronic acid-functionalized quinone-cyanine (QCy-BA) in combination with AT-rich DNA (exogenous or endogenous cellular DNA), i.e., QCy-BA subset of DNA as a stimuli-responsive NIR fluorescence probe for measuring in vitro levels of H2O2. In response to cellular H2O2 stimulus, QCy-BA converts into QCy-DT, a one-donor-two-acceptor (D2A) system that exhibits switch-on NIR fluorescence upon binding to the DNA minor groove. Fluorescence studies on the combination probe QCy-BA subset of DNA showed strong NIR fluorescence selectively in the presence of H2O2. Furthermore, glucose oxidase (GOx) assay confirmed the high efficiency of the combination probe QCy-BA subset of DNA for probing H2O2 generated in situ through GOx-mediated glucose oxidation. Quantitative analysis through fluorescence plate reader, flow cytometry and live imaging approaches showed that QCy-BA is a promising probe to detect the normal as well as elevated levels of H2O2 produced by EGF/Nox pathways and post-genotoxic stress in both primary and senescent cells. Overall, QCy-BA, in combination with exogenous or cellular DNA, is a versatile probe to quantify and image H2O2 in normal and disease-associated cells.

Thermodynamics of ligand (substrate end product) binding to endoxylanase from Chainia sp. (NCL-82-5-1): isothermal calorimetry and fluorescence titration studies

The binding of xylo-oligosaccharides to Chainia endoxylanase resulted in a decrease in fluorescence intensity of the enzyme with the formation of 1:1 complex. Equilibrium and thermodynamic parameters of ligand binding were determined by fluorescence titrations and titration calorimetry. The affinity of xylanase for the oligosaccharides increases in the order X-2 < X-3 < X-4 less than or equal to X-5. Contributions from the enthalpy towards the free energy change decreased with increasing chain length from X-2 to X-4, whereas an increase in entropy was observed, the change in enthalpy and entropy of binding being compensatory. The entropically driven binding process suggested that hydrophobic interactions as well as hydrogen bonds play a predominant role in ligand binding.

Estimation of mineral-adhered biomass of Thiobacillusferrooxidans by protein assay-some problems and remedies

Enumeration of adhered cells of Thiobacillus ferrooxidans on sulphide minerals through protein assay poses problems due to interference from dissolved mineral constituents. The manner in which sulphide minerals such as pyrite, chalcopyrite, sphalerite, arsenopyrite and pyrrhotite interfere with bacterial protein estimation is demonstrated. Such interferences can be minimised either through dilution or addition of H2O2 to the filtrate after hot alkaline digestion of the biotreated mineral samples.

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.