Electrochemical current rectifier as a highly sensitive and selective cytosensor for cancer cell detection

Signal amplification originating from electrochemical current rectifier (ECR) was firstly applied to construct a cytosensor for rapid and non-invasive detection of folate receptor-rich cancer cells with high sensitivity. It exhibits a broad linear range with a detection limit as low as 10 cells mL(-1) even in the presence of a large number of normal cells.     

Biosensor incorporating cell barrier architectures on ion selective electrodes for early screening of cancer

Angiogenesis occurs during the early phase of cancer. Recruitment of new blood vessels by existing cancer cells leads to the release of higher concentrations of cytokines as compared to cells in healthy individuals. Some of the common cytokines observed at higher concentrations, such as vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and tumor necrosis factor-alpha, are also known to induce increased permeability across an endothelial cell monolayer. A whole-cell-based biosensor has been developed that can detect the presence of small quantities of the abovementioned cytokines individually and in different combinations. It was observed that the biosensor could differentiate between the cytokine concentrations observed in the sera of healthy individuals and cancer patients. The biosensor was also evaluated by exposing it to actual serum. These results demonstrated that the sensor can distinguish between healthy individuals and cancer patients and that the corresponding biosensor responses correlate with the stages of cancer.     

Characterization of newly established oral cancer cell lines derived from six squamous cell carcinoma and two mucoepidermoid carcinoma cells

Since genetic abnormalities of human cancer are greatly geographically dependent, cultural and environmental backgrounds are thought to be closely related to the carcinogenic process. In the present study, eight human cell lines were established by culture from untreated carcinomas of the oral cancer, of which five were from primary oral squamous cell carcinomas (OSC), one from a mucoepidermoid carcinoma (MEC) and one each originating from metastatic OSC and MEC. All the studied tumor lines grew as monolayers, and showed: i) an epithelial origin by the presence of cytokeratin, and ii) tumorigenic potential in nude mice. Western blot analysis revealed i) over expression of EGFR in six of the cell lines ii) decreased expression of E-cadherin in six cell lines compared to normal human oral mucosa. A mutational analysis showed: point mutations of p53 at exon 7, with transversion, and at exon 8, with transition. These well-characterized human YD cell lines should serve as useful tools in the study of the molecular pathogenesis and biological characteristics of head and neck cancer cells, and in the future testing of new therapeutic reagents for oral cancer.     

4-azidoquinoline N-oxide: Synthesis and photolysis

In photolysis of 4-azidoquinoline N-oxide azoxy compound was isolated as the final reaction product. This result may be ascribed to the dimerization of the intermediate nitrene to azo compound followed by oxidation of the latter with air oxygen. The initially arising nitrene is stabilized by resonance conjugation involving the aromatic system and the N-oxide group. The rate constants of 4-azidoquinoline N-oxide photolysis were measured in various solvents and the values of spin density and bond lengths in the formed nitrene were calculated.     

Porphyrin-bile acid conjugates: from saccharide recognition in the solution to the selective cancer cell fluorescence detection

This paper describes the preparation and use of conjugates of porphyrins and bile acids as ligands to bind to tumor expressed saccharides. Bile acid-porphyrin conjugates were tested for recognition of saccharides that are typically present on malignant tumor cells. Fluorescence microscopy, in vitro PDT cell killing, and PDT of subcutaneous 4T1 mouse tumors is reported. High selectivity for saccharide cancer markers and cancer cells was observed. This in vivo and in vitro study demonstrated high potential use for these compounds in targeted photodynamic therapy.     

Enhanced photolysis in aerosols: evidence for important surface effects

While there is increasing evidence for unique chemical reactions at interfaces, there are fewer data on photochemistry at liquid-vapor junctions. This paper reports a comparison of the photolysis of molybdenum hexacarbonyl, Mo(CO)(6), in 1-decene either as liquid droplets or in bulk-liquid solutions. Mo(CO)(6) photolysis is faster by at least three orders of magnitude in the aerosols than in bulk-liquids. Two possible sources of this enhancement are considered: (1) increased light intensity due to either Morphology-Dependent Resonances (MDRs) in the spherical aerosol particles and/or to increased pathlengths for light inside the droplet due to refraction, which are termed physical effects in this paper; and (2) interface effects such as an incomplete solvent-cage at the gas-liquid boundary and/or enhanced interfacial concentrations of Mo(CO)(6), which are termed chemical effects. Quantitative calculations of the first possibility were carried out in which the light intensity distribution in the droplets averaged over 215-360 nm was obtained for 1-decene droplets. Calculations show that the average increase in light intensity over the entire droplet is 106%, with an average increase of 51% at the interface. These increases are much smaller than the observed increase in the apparent photolysis rate of droplets compared to the bulk. Thus, chemical effects, i.e., a decreased solvent-cage effect at the interface and/or enhancement in the surface concentration of Mo(CO)(6), are most likely responsible for the dramatic increase in the photolysis rate. Similar calculations were also carried out for broadband (290-600 nm) solar irradiation of water droplets, relevant to atmospheric conditions. These calculations show that, in agreement with previous calculations by Mayer and Madronich [B. Mayer and S. Madronich, Atmos. Chem. Phys., 2004, 4, 2241] MDRs produce only a moderate average intensity enhancement relative to the corresponding bulk-liquid slabs when averaged over a range of wavelengths characteristic of solar radiation at the Earth's surface. However, as in the case of Mo(CO)(6) in 1-decene, chemical effects may play a role in enhanced photochemistry at the aerosol-air interface for airborne particles.     

Experimental and theoretical study of two-photon absorption in nitrofuran derivatives: Promising compounds for photochemotherapy

We report experimental and theoretical studies of the two-photon absorption spectrum of two nitrofuran derivatives: nitrofurantoine, (1-(5-nitro-2-furfurilideneamine)-hidantoine) and quinifuryl, 2-(5(')-nitro-2(')-furanyl)ethenyl-4-{N-[4(')-(N,N-diethylamino)-1(')-methylbutyl]carbamoyl} quinoline. Both molecules are representative of a family of 5-nitrofuran-ethenyl-quinoline drugs that have been demonstrated to display high toxicity to various species of transformed cells in the dark. We determine the two-photon absorption cross-section for both compounds, from 560 to 880 nm, which present peak values of 64 GM for quinifuryl and 20 GM for nitrofurantoine (1 GM = 1×10(-50)cm(4).s.photon(-1)). Besides, theoretical calculations employing the linear and quadratic response functions were carried out at the density functional theory level to aid the interpretations of the experimental results. The theoretical results yielded oscillator strengths, two-photon transition probabilities, and transition energies, which are in good agreement with the experimental data. A higher number of allowed electronic transitions was identified for quinifuryl in comparison to nitrofurantoine by the theoretical calculations. Due to the planar structure of both compounds, the differences in the two-photon absorption cross-section values are a consequence of their distinct conjugation lengths.     

Strategies for kinome profiling in cancer and potential clinical applications: chemical proteomics and array-based methods

Kinases are key enzymes involved in deregulated signal transduction associated with cancer development and progression. The advent of personalized medicine drives the development of new diagnostic tools for patient stratification and therapy selection Ginsburg and Willard (Transl Res 154:277-287, 2009). Since deregulation of kinase-mediated signal transduction is implied in tumorigenesis, the analysis of all kinases (the kinome) active in a particular tumor may yield tumor-specific information on aberrant cell signalling pathways. Tumor tissue kinase activity profiles may correlate with response to therapy and therefore may be used for future therapy selection. In this Trend paper we describe peptide array and mass spectrometry-based technologies and new developments for kinome profiling, and we present an outlook towards future implementation of therapy selection based on kinome profiling in clinical practice.     

Diamino acid derivatives of PpIX as potential photosensitizers for photodynamic therapy of squamous cell carcinoma and prostate cancer: In vitro studies

Photodynamic therapy (PDT) is an alternative treatment modality involving light activated drugs, called photosensitizers (PSs), to treat cancer and non-cancerous conditions. The search for new compounds which might become effective PSs is the major direction for PDT development. In the present work we have studied the dark toxicity, intracellular localization and photodynamic properties of four potential, water soluble, second generation PSs – PP(Arg)₂, PP(Ser)₂Arg₂, PP(Ala)₂Arg₂, PP(Phe)₂Arg₂, all diamino acid derivatives of protoporphyrin IX. Human prostate cancer (DU-145) and squamous carcinoma (A431) cells were used as experimental model.Among investigated compounds PP(Ser)₂Arg₂ exhibited the lowest dark toxicity and the highest PDT effectiveness towards both cell lines. Fluorescence microscopy revealed the time-dependent changes in intracellular localization of the PS which were related to the phototoxicity. The results show that PP(Ser)₂Arg₂ may be a potential PS for PDT.     

Fluorescence contrast and threshold limit: implications for photodynamic diagnosis of basal cell carcinoma

This study was designed to evaluate what application time of delta-5-aminolaevulinic acid (ALA) results in highest contrast between tumour and normal skin, in the interval 1-4 h, when using photodynamic diagnosis (PDD) of basal cell carcinomas (BCC) located on the face. Moreover, a value of the demarcation limit has been derived based on the fluorescence variation in normal skin adjacent to the tumour. Forty patients were included in the study, randomly allocated to four different groups with varying ALA application time in the range 1-4 h. The contrast, defined as the ratio between the fluorescence intensity in ALA-treated tumour tissue and normal skin, was calculated for each patient, and the mean values in each group were evaluated as a function of ALA application time. In addition, the fluorescence intensity variation in ALA-treated normal skin adjacent to the tumour was assessed. The results from this study show a peak of the mean contrast values after 3 h ALA application, but due to large interpatient variation, the mean contrast did not differ significantly in the interval 2-4 h. After 2 h ALA application, the fluorescence intensity variation in the normal ALA-treated skin was found to be at a maximum, which suggests that 2 h ALA application is not preferable when using PDD. Based on data of the fluorescence variation in ALA-treated normal skin after 3 and 4 h ALA application, a tolerance interval was calculated implying that values above 1.4 times the mean normal fluorescence indicate an abnormal condition. This tolerance limit agrees well with results obtained in a former study.     

The construction of an individually addressable cell array for selective patterning and electroporation

In basic cell biology research and drug discovery, it is important to rapidly introduce genes, proteins or drug compounds into cells without permanent damage. Here, we report a three dimensional SU-8 micro-well structure sandwiched with an indium tin oxide (ITO) electrode-covered slide from the top and an individually addressable array of microelectrodes on the bottom to allow parallel delivery of exogenous molecules into various cells in a spatially specific manner. A positive dielectrophoretic force was selectively applied by energizing appropriate electrodes to capture the dispersed cells at the bottom electrode, while the micro-wells were designed to confine cells in situ when the positive dielectrophoretic force is removed. The combination of spatial positive dielectrophoresis (pDEP) and micro-wells made it possible to construct cell microarrays with specific patterns. Once the cells become attached to the electrodes, different plasmids can be introduced sequentially for selective electroporation. The present cell arraying-assisted electroporation chip integrates a pDEP-assisted cell positioning function with selective electroporation to provide a simple and efficient method for gene transfer. This platform is ideal for high throughput screening of compounds in parallel and thus holds promise for applications in cellular and molecular research.     

Gamma radiolysis and solar photolysis of bilirubin: Similarities and differences

Biliverdin is a useful component in various aspects of biochemistry and biosynthesis, but its synthetic preparation is often long-winded. Micro-production (and subsequent isolation) by solar photolysis and gamma radiolysis of bilirubin provides rapid in vitro generation. Both methods are competitive, and this article discusses their merits and limitations for application in biosynthetic research. The investigation assumed a comparative study to evaluate the relative potential of the photolytic and radiolytic phenomena in this respect. The calculated rate of incident energy in the case of solar photolysis was roughly30.4^.10^-2 W, and about 5.70^.10^-4 W during gamma-irradiation (from a ¹³⁷Cs source). In both cases the bilirubin (40 μM) degradation was pronounced in the initial few minutes of exposure, producing respective depletion rates of approximately 6.8 μM/min and 2.4 μM/min. Overall, both applications showed declining bilirubin concentrations close to 90%, after about 30 minutes. However, the corresponding production of biliverdin was higher by about 50% in the photolytic application. To account for heat-up effects in the photolytic application, thermal effects were studied up to 65 °C, and it was found that, as a result of this, a reduction in bilirubin concentration of about 40% was encountered. The species of interest were monitored spectrophotometrically, and the composite results showed that regulated production of biliverdin is possible under certain conditions. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phenacyl ester derivatives bearing heterocycles as models for photocleavable linkers: synthesis and photolysis studies

The synthesis of several phenacyl ester derivatives linked to oxygen and nitrogen heterocycles, benzoquinolone and (benzo)coumarins, was carried out in an effort to obtain systems that could be applied as photocleavable (bi)functional linkers for solid phase peptide synthesis. The heterocycles were attached to a spacer, with different lengths, followed by coupling to 2-bromo-1-phenylpropan-1-one, acting as a model for the solid support. Photolysis studies of the resulting phenacyl ester derivatives were carried out by irradiation in a photochemical reactor at different wavelengths (300, 350 and 419 nm), in methanol/HEPES buffer solution (80:20).     

Spectral and kinetic parameters of transient species in the photolysis of naphthylmethylideneiminospironaphthopyran by excitation at different wavelengths: Nano- and femtosecond laser photolysis

Intramolecular processes occurring in a photobifunctional compound (PBC) comprising the spironaphthopyran and hydroxyazomethine moieties have been studied in methanol solutions by femtosecond laser photolysis using light with wavelengths of 340 and 490 nm. At the excitation wavelength of 490 nm, the cis-trans photoisomerization in the azomethine moiety occurs in the S₁ state. In the case of PBC photolysis with 340-nm light, the opening of the spiro bond of the spiropyran moiety (formation of the X form) also takes place during relaxation of the S_n state to the S₁ state followed by isomerization to the merocyanine form. The spectral and kinetic characteristics of different electronically excited have been were determined. The data have been compared with those of nanosecond laser photolysis.     

Cleavage of carbon-carbon bonds of diphenylacetylene and its derivatives via photolysis of Pt complexes: tuning the C-C bond formation energy toward selective C-C bond activation

Carbon-carbon bond activation of diphenylacetylene and several substituted derivatives has been achieved via photolysis and studied. Pt0-acetylene complexes with eta2-coordination of the alkyne, along with the corresponding PtII C-C activated photolysis products, have been synthesized and characterized, including X-ray crystal structural analysis. While the C-C cleavage reaction occurs readily under photochemical conditions, thermal activation of the C-C bonds or formation of PtII complexes was not observed. However, the reverse reaction, C-C reductive coupling (PtII --> Pt0), did occur under thermal conditions, allowing the determination of the energy barriers for C-C bond formation from the different PtII complexes. For the reaction (dtbpe)Pt(-Ph)(-CCPh) (2) --> (dtbpe)Pt(eta2-PhCCPh) (1), DeltaG was 32.03(3) kcal/mol. In comparison, the energy barrier for the C-C bond formation in an electron-deficient system, that is, (dtbpe)Pt(C6F5)(CCC6F5) (6) --> (dtbpe)Pt(eta2-bis(pentafluorophenyl)acetylene) (5), was found to be 47.30 kcal/mol. The energy barrier for C-C bond formation was able to be tuned by electronically modifying the substrate with electron-withdrawing or electron-donating groups. Upon cleavage of the C-C bond in (dtbpe)Pt(eta2-(p-fluorophenyl-p-tolylacetylene) (9), both (dtbpe)Pt(p-fluorophenyl)(p-tolylacetylide) (10) and (dtbpe)Pt(p-tolyl)(p-fluorophenylacetylide) (11) were obtained. Kinetic studies of the reverse reaction confirmed that 10 was more stable toward the reductive coupling [the term "reductive coupling" is defined as the formation of (dtbpe)Pt(eta2-acetylene) complex from the PtII complex] than 11 by 1.22 kcal/mol, under the assumption that the transition-state energies are the same for the two pathways. The product ratio for 10 and 11 was 55:45, showing that the electron-deficient C-C bond is only slightly preferentially cleaved.     

Strategies for the purification of laminin-10 for studies on colon cancer metastasis

Signals from the epidermal growth factor (EGF) receptor family are thought to combine with integrin-dependent adhesion to laminins to contribute to disease progression and metastasis in cancer. To date, little is known about the mechanisms by which these signals interact. Recently, we have shown that the colon cancer cell line LIM1215 secretes and adheres to laminin-10 through multiple integrin receptors, and that EGF stimulates spreading and migration of these cells on the same substrate. Additionally laminin-10/11 has been shown by immunohistochemistry to be present at the invasive edge of moderately differentiated colon cancers. To enable detailed structure-function studies to be undertaken, it is important to be able to rapidly obtain highly purified native laminin-10 from bulk biological samples in reasonable yield. The development of a multidimensional micropurification scheme to achieve this is presented and compared with other reported methods for the purification of laminins.     

Molecular-modulation spectrometry: CH3ONO photolysis and detection of nitroxyl

The method of molecular-modulation spectrometry for studying photochemical reactions has been applied to methyl nitrite photolysis. The infrared absorption of the nitroxyl radical HNO has been observed in the gas phase at 3300 cm^?1. Under the present experimental conditions the steady-state concentration of HNO under steady illumination was 1.1 × 10¹² particles/cc, and the observed modulation amplitude was 4.5 × 10¹⁰ particles/cc. At 25°C and 1 atm of nitrogen, the cross section for infrared absorption by HNO at 3300 cm^?1 is 1.7 × 10^?19 cm². The rate constant ratio b/c was found to be 8.0. From the literature value of the rate constant d , the observed rate constant for the reaction is e = (5 ± 1) × 10^?11 cc/particle sec.     

Photochemistry of 5-allyloxy-tetrazoles: steady-state and laser flash photolysis study

The photochemistry of three 5-allyloxy-tetrazoles, in methanol, acetonitrile and cyclohexane was studied by product analysis and laser flash photolysis. The exclusive primary photochemical process identified was molecular nitrogen elimination, with formation of 1,3-oxazines. These compounds were isolated in reasonable yields by column chromatography on silica gel and were fully characterized. DFT(B3LYP)/6-31G(d,p) calculations predict that these 1,3-oxazines can adopt two tautomeric forms (i) with the NH group acting as a bridge connecting the oxazine and phenyl rings and (ii) with the -N=bridge and the proton shifted to the oxazine ring. Both tautomeric forms are relevant in the photolysis of oxazines in solution. Secondary reactions were observed, leading to the production of phenyl vinyl-hydrazines, enamines, aniline and phenyl-isocyanate. Transient absorption, detected by laser flash photolysis, is attributed to the formation of triplet 1,3-biradicals generated from the excited 5-allyloxy-tetrazoles. The 1,3-biradicals are converted to 1,6-biradicals by proton transfer, which, after intersystem crossing, decay to generate the products. Solvent effects on the photoproduct distribution and rate of decomposition are negligible.     

A naphthalene-based Al3+ selective fluorescent sensor for living cell imaging

An efficient fluorescent Al(3+) receptor, N-(2-hydroxy-1-naphthalene)-N'-(2-(2-hydroxy-1-naphthalene)amino-ethyl)-ethane-1,2-diamine (L) has been synthesized by the condensation reaction between 2-hydroxy naphthaldehyde and diethylenetriamine. High selectivity and affinity of L towards Al(3+) in ethanol (EtOH) as well as in HEPES buffer at pH 7.4, makes it suitable to detect intracellular Al(3+) with fluorescence microscopy. Metal ions, viz. Li(+), Na(+), K(+), Mg(2+), Ca(2+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Ag(+), Cd(2+), Hg(2+) and Pb(2+) do not interfere. The lowest detection limit for Al(3+) is 3.0 × 10(-7) M and 1.0 × 10(-7) M in EtOH and HEPES buffer respectively.