RESUMO
The migration of an electron-loss center (hole) in calf thymus DNA to bisbenzimidazole ligands bound in the minor groove is followed by pulse radiolysis combined with time-resolved spectrophotometry. The initially observed absorption spectrum upon oxidation of DNA by the selenite radical is consistent with spin on cytosine (C), as the GC⢠pair neutral radical, followed by the spectra of oxidized ligands. The rate of oxidation of bound ligands increased with an increase in the ratio (r) ligands per base pair from 0.005 to 0.04. Both the rate of ligand oxidation and the estimated range of hole transfer (up to 30 DNA base pairs) decrease with the decrease in one-electron reduction potential between the GC⢠pair neutral radical of ca. 1.54 V and that of the ligand radicals (E0', 0.90-0.99 V). Linear plots of log of the rate of hole transfer versus r give a common intercept at r = 0 and a free energy change of 12.2 ± 0.3 kcal mol-1, ascribed to the GC⢠pair neutral radical undergoing a structural change, which is in competition to the observed hole transfer along DNA. The rate of hole transfer to the ligands at distance, R, from the GC⢠pair radical, k2, is described by the relationship k2 = k0 exp(constant/R), where k0 includes the rate constant for surmounting a small barrier.
Assuntos
Pareamento de Bases , DNA , DNA/química , Radicais Livres/química , Oxirredução , Benzimidazóis/química , Animais , Bovinos , Ligantes , Bisbenzimidazol/química , Reparo do DNA , Dano ao DNA , Citosina/químicaRESUMO
Although DNA-ligand binding is pervasive in biology, little is known about molecular-level binding mechanisms. Using all-atom, explicit-solvent molecular dynamics simulations in conjunction with weighted ensemble (WE)-enhanced sampling, an ensemble of 2562 binding trajectories of Hoechst 33258 (H33258) to d(CGC AAA TTT GCG) was generated from which the binding mechanism was extracted. In particular, the electrostatic interaction between the positively charged H33258 and the negatively charged DNA backbone drives the formation of initial H33258-DNA contacts. After this initial contact, a hinge-like intermediate state is formed in which one end of H33258 inserts into the minor groove of DNA. Following hinge state formation is a concerted motion whereby the second end of H33258 swings into the minor groove and the spine of hydration along the minor groove causing dehydration. This study illustrates how WE-enhanced simulations of biomolecular ligation processes can offer novel mechanistic insights by generating ensembles of binding events.
Assuntos
Bisbenzimidazol , DNA , Conformação de Ácido Nucleico , Ligantes , DNA/química , Bisbenzimidazol/químicaRESUMO
The interaction between oral contraceptive drug Ormeloxifene (ORM) and calf thymus DNA (CT-DNA) was studied using UV-Vis, fluorescence, circular dichroism (CD) and 1H NMR spectral techniques under physiological buffer (pH 7.4). Competitive binding assays with ethidium bromide (EB) and Hoechst 33258, viscosity measurements, KI quenching studies, molecular docking and metadynamics simulation studies were also substantiated the spectroscopic results. ORM is found to binds in the minor groove of CT-DNA as evidenced by: (1) non-displacement of EB from EB/CT-DNA complex; (2) appreciable displacement of Hoechst 33258 from its CT-DNA complex; (3) slight alteration in the CD signal; (4) small shifts (Δδ < 0.033 ppm) without broadening in 1H NMR signals and (5) the nearly equal extent of quenching of fluorescence of ORM by KI in the absence and presence of CT-DNA. Negative values of both enthalpy and entropy changes pointed out that the interaction between ORM and CT-DNA is governed mainly by H-bonding and van der Waals forces. Negative free energy change suggested a spontaneous interaction between ORM and CT-DNA. The free energy landscape of the binding process was computed using metadynamics simulation. The simulation study results disclosed that ORM binds to the minor groove of DNA through H-bonding and π-π stacking interactions. The results of molecular docking and simulation studies corroborate the available experimental data.
Assuntos
Bisbenzimidazol , DNA , Benzopiranos , Bisbenzimidazol/química , Dicroísmo Circular , Anticoncepcionais Orais , DNA/química , Feminino , Humanos , Simulação de Acoplamento Molecular , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , Termodinâmica , Tomografia Computadorizada por Raios XRESUMO
The shortcomings of current anti-human cytomegalovirus (HCMV) drugs has stimulated a search for anti-HCMV compounds with novel targets. We screened collections of bioactive compounds and identified a range of compounds with the potential to inhibit HCMV replication. Of these compounds, we selected bisbenzimide compound RO-90-7501 for further study. We generated analogues of RO-90-7501 and found that one compound, MRT00210423, had increased anti-HCMV activity compared to RO-90-7501. Using a combination of compound analogues, microscopy and biochemical assays we found RO-90-7501 and MRT00210423 interacted with DNA. In single molecule microscopy experiments we found RO-90-7501, but not MRT00210423, was able to compact DNA, suggesting that compaction of DNA was non-obligatory for anti-HCMV effects. Using bioinformatics analysis, we found that there were many putative bisbenzimide binding sites in the HCMV DNA genome. However, using western blotting, quantitative PCR and electron microscopy, we found that at a concentration able to inhibit HCMV replication our compounds had little or no effect on production of certain HCMV proteins or DNA synthesis, but did have a notable inhibitory effect on HCMV capsid production. We reasoned that these effects may have involved binding of our compounds to the HCMV genome and/or host cell chromatin. Therefore, our data expand our understanding of compounds with anti-HCMV activity and suggest targeting of DNA with bisbenzimide compounds may be a useful anti-HCMV strategy.
Assuntos
Antivirais/farmacologia , Bisbenzimidazol/farmacologia , Citomegalovirus/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Antivirais/química , Sítios de Ligação , Bisbenzimidazol/química , Capsídeo/metabolismo , Linhagem Celular , Citomegalovirus/fisiologia , DNA/biossíntese , DNA/química , Replicação do DNA/efeitos dos fármacos , Humanos , Estrutura Molecular , Carga Viral/efeitos dos fármacosRESUMO
The versatility of DNA minor groove binding bibenzimidazoles extends to applications in cancer therapy, beyond their typical use as DNA stains. In the context of UVA phototherapy, a series of halogenated analogues designated ortho-, meta-, and para-iodoHoechst have been investigated. Phototoxicity involves dehalogenation of the ligands following exposure to UVA light, resulting in the formation of a carbon-centred radical. While the cytotoxic mechanisms have been well established, the nature and severity of DNA damage induced by the ortho-, meta-, and para-iodoHoechst isomers requires clarification. Our aims were to measure and compare the binding constants of iodoHoechst analogues, and to determine the proximity of the carbon-centred radicals formed following photodehalogenation to the C1', C4', and C5' DNA carbons. We performed molecular docking studies, as well as classical molecular dynamics simulations to investigate the interactions of Hoechst ligands with DNA including a well-defined B-DNA dodecamer containing the high affinity AATT minor groove binding site. Docking highlighted the binding of Hoechst analogues to AATT regions in oligonucleotides, nucleosomes, and origami DNA helical bundles. Further, MD simulations demonstrated the stability of Hoechst ligands in the AATT-containing minor groove over microsecond trajectories. Our findings reiterate that the efficiency of dehalogenation per se, rather than the proximity of the carbon-centred radicals to the DNA backbone, is responsible for the extreme phototoxicity of the ortho- isomer compared to the meta- and para-iodoHoechst isomers. More generally, our analyses are in line with the potential utility of ortho-iodoHoechst in DNA-targeted phototherapy, particularly if combined with a cell-specific delivery system.
Assuntos
Bisbenzimidazol/química , DNA/química , Simulação de Acoplamento Molecular , Sítios de LigaçãoRESUMO
Changes in the structural dynamics of double stranded (ds)DNA upon ligand binding have been linked to the mechanism of allostery without conformational change, but direct experimental evidence remains elusive. To address this, a combination of steady state infrared (IR) absorption spectroscopy and ultrafast temperature jump IR absorption measurements has been used to quantify the extent of fast (â¼100 ns) fluctuations in (ds)DNA·Hoechst 33258 complexes at a range of temperatures. Exploiting the direct link between vibrational band intensities and base stacking shows that the absolute magnitude of the change in absorbance caused by fast structural fluctuations following the temperature jump is only weakly dependent on the starting temperature of the sample. The observed fast dynamics are some two orders of magnitude faster than strand separation and associated with all points along the 10-base pair duplex d(GCATATATCC). Binding the Hoechst 33258 ligand causes a small but consistent reduction in the extent of these fast fluctuations of base pairs located outside of the ligand binding region. These observations point to a ligand-induced reduction in the flexibility of the dsDNA near the binding site, consistent with an estimated allosteric propagation length of 15 Å, about 5 base pairs, which agrees well with both molecular simulation and coarse-grained statistical mechanics models of allostery leading to cooperative ligand binding.
Assuntos
DNA/química , Sítio Alostérico , Pareamento de Bases , Sequência de Bases , Bisbenzimidazol/química , Cinética , Ligantes , Modelos Moleculares , Conformação de Ácido Nucleico , Espectrofotometria Infravermelho , TemperaturaRESUMO
At present, nuclear condensation and fragmentation have been estimated also using Hoechst probes in fluorescence microscopy and flow cytometry. However, none of the methods used the Hoechst probes for quantitative spectrofluorometric assessment. Therefore, the aim of the present study was to develop a spectrofluorometric assay for detection of nuclear condensation and fragmentation in the intact cells. We used human hepatoma HepG2 and renal HK-2 cells cultured in 96-well plates treated with potent apoptotic inducers (i.e. cisplatin, staurosporine, camptothecin) for 6-48 h. Afterwards, the cells were incubated with Hoechst 33258 (2 µg/mL) and the increase of fluorescence after binding of the dye to DNA was measured. The developed spectrofluorometric assay was capable to detect nuclear changes caused by all tested apoptotic inducers. Then, we compared the outcomes of the spectrofluorometric assay with other methods detecting cell impairment and apoptosis (i.e. WST-1 and glutathione tests, TUNEL, DNA ladder, caspase activity, PARP-1 and JNKs expressions). We found that our developed spectrofluorometric assay provided results of the same sensitivity as the TUNEL assay but with the advantages of being fast processing, low-cost and a high throughput. Because nuclear condensation and fragmentation can be typical markers of cell death, especially in apoptosis, we suppose that the spectrofluorometric assay could become a routinely used method for characterizing cell death processes.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Espectrometria de Fluorescência/métodos , Bisbenzimidazol/química , Camptotecina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/metabolismo , Cisplatino/farmacologia , Citometria de Fluxo/métodos , Células Hep G2 , Humanos , Microscopia de Fluorescência/métodos , Reprodutibilidade dos Testes , Estaurosporina/farmacologiaRESUMO
Alicyclobacillus acidoterrestris is an acidophilic and thermophilic bacterium present in the soil, often associated with the spoilage of acidic juices, such as orange juice. Their spores resist pasteurization and, when reactivated, modify the organoleptic properties of the juice, making it unsuitable for consumption, due mainly to production of guaiacol. Biosensors are detection devices that respond quickly and are easy to handle, with great potential for use in the juice production chain. In this context, this work reports an electrochemical genosensor for detection of A. acidoterrestris, based on a graphite electrode modified with electrochemically reduced graphene oxide, a polymer derived from 3-hydroxybenzoic acid and a specific DNA probe sequence complementary with the genomic DNA of A. acidoterrestris. Detection of the target was performed by monitoring the oxidation peak of the Hoechst 33258, a common DNA stainer. The genosensor detection limit was 12 ng mL-1 and it kept 77% of response after ten weeks, and a test showed that orange juice does not interfere with bacteria lysate detection. This biosensor is the first platform for electrochemical detection of the genomic DNA of A. acidoterrestris in the literature, and the first to use Hoechst 33258 as indicator with whole genomic DNA molecules.
Assuntos
Alicyclobacillus/isolamento & purificação , Técnicas Biossensoriais/métodos , Bisbenzimidazol/química , DNA Bacteriano/análise , DNA Bacteriano/química , Alicyclobacillus/genética , Eletroquímica , Eletrodos , Grafite/química , OxirreduçãoRESUMO
A set of AT-specific fluorescent dimeric bisbenzimidazoles DBPA(n) with linkers of different lengths bound to DNA in the minor groove were synthesized and their genetic, virological, and biochemical studies were performed. The DBPA(n) were shown to be effective inhibitors of the histon-like protein H-NS, a regulator of the DNA transcription factor, as well as of the Aliivibrio logei Quorum Sensing regulatory system in E. coli cells. Their antiviral activity was tested in model cell lines infected with herpes simplex virus type I. Also, it was found that DBPA(n) could inhibit catalytic activities of HIV-1 integrase at low micromolar concentrations. All of the dimeric bisbenzimidazoles DBPA(n) manifested fluorescent properties, were well soluble in water, nontoxic up to concentrations of 200 µM, and could penetrate into nuclei followed by binding to DNA.
Assuntos
Bisbenzimidazol/química , Bisbenzimidazol/farmacologia , DNA/química , Aliivibrio/metabolismo , Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Sequência de Bases , DNA/genética , Desenho de Fármacos , Escherichia coli/metabolismo , Corantes Fluorescentes , Integrase de HIV , Inibidores de Integrase de HIV/farmacologia , Ligantes , Estrutura Molecular , Pirróis , Percepção de Quorum/fisiologia , Relação Estrutura-AtividadeRESUMO
Nitrogen-containing heterocycles are one of the most common structural motifs in approximately 80% of the marketed drugs. Of these, benzimidazoles analogues are known to elicit a wide spectrum of pharmaceutical activities such as anticancer, antibacterial, antiparasitic, antiviral, antifungal as well as chemosensor effect. Based on the benzimidazole core fused heterocyclic compounds, crescent-shaped bisbenzimidazoles were developed which provided an early breakthrough in the sequence-specific DNA recognition. Over the years, a number of functional variations in the bisbenzimidazole core have led to the emergence of their unique properties and established them as versatile ligands against several classes of pathogens. The present review provides an overview of diverse pharmacological activities of the bisbenzimidazole analogues in the past decade with a brief account of its development through the years.
Assuntos
Bisbenzimidazol/farmacologia , Descoberta de Drogas , Bisbenzimidazol/química , HumanosRESUMO
DNA strands can be designed to assemble into stable three-dimensional structures, based on Watson-Crick base pairing rules. The simplest of these is the DNA tetrahedron that is composed of four oligonucleotides. We have re-designed the sequence of a DNA tetrahedron so that it contains a single (AATT) binding site for the minor groove binding ligand Hoechst 33258. We examined the stability of this structure by placing fluorescent groups within each of its edges and have shown that all the edges melt at the same temperature in the absence of the ligand. The minor groove ligand still binds to its recognition sequence within the tetrahedron and increases the melting temperature of the folded complex. This ligand-induced stabilisation is propagated into the adjacent helical arms and the tetrahedron melts as a single entity in a cooperative fashion.
Assuntos
DNA/química , Ligantes , Sequência de Bases , Sítios de Ligação , Bisbenzimidazol/química , Bisbenzimidazol/metabolismo , Conformação de Ácido Nucleico , Transição de Fase/efeitos da radiação , Espectrometria de Fluorescência , Temperatura de Transição , Raios UltravioletaRESUMO
Antibiotic residues can induce the production of bacterial variability and increase the variety of resistant bacterial strains. In order to cope up with the global crisis, there is an urgent need to develop alternative drugs and strategies to effectively kill the bacteria and inhibit the emergence of superbugs. Herein, we developed an antibacterial strategy based on the conjugated polymer nanoparticles (CPNs) and the biomolecule-mediated regulation for the controllable antibacterial treatment. Upon being modified with Hoechst 33258 (H33258), the biocompatible CPNs tend to be effective antibacterial agents, with the antibacterial activity being regulated through the binding and separation of H33258 and calf thymus double-stranded DNA (dsDNA) with the aid of deoxyribonuclease I (DNase I). This eco-friendly antibacterial system not only provides a simple and controllable antibacterial strategy but also inspires the design of biomolecule-mediated antibacterial activity regulation.
Assuntos
Antibacterianos/administração & dosagem , Bisbenzimidazol/química , Infecções por Escherichia coli/tratamento farmacológico , Polímeros/administração & dosagem , Animais , Antibacterianos/síntese química , Antibacterianos/química , Antibacterianos/farmacologia , DNA/efeitos dos fármacos , Modelos Animais de Doenças , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Feminino , Química Verde , Camundongos , Nanoconchas , Polímeros/síntese química , Polímeros/química , Polímeros/farmacologiaRESUMO
A key utility of fluorophores lies in sensing applications: the detection of changes to emission caused by differences in their microenvironment. The rational design of fluorescent sensors remains a significant challenge because of the complexity of factors which control molecular deactivation pathways. Here, in an effort to define the structural criteria underlying the fluorescence turn-on response of Hoechst 33258 (H33258) upon binding to the DNA minor groove, we examine this sensor's intrinsic properties in minimalist microenvironments. We first characterised the intrinsic photophysics of gaseous mono- and di-protonated H33258 ions, then introduced intermolecular interactions by complexation with double-stranded (ds) DNA. Selected-ion laser-induced fluorescence (SILIF) and photodissociation of the gaseous monoprotomers indicate the presence of multiple populations with distinct fluorescence and dissociation properties. We assign one of these to a kinetically-trapped form which is protonated at the site favored in solution. The other form exhibits a more intense emission band which is shifted by more than 6000 cm-1 to the red of the first form. Quantum chemical calculations reveal that this second population is likely a newly-identified protomer, which is considerably more stable in the gas phase than conformations with the solution protonation site. Two routes that increase the fluorescence of H33258 in solution - formation of the diprotomer and complexation with dsDNA - do not produce an increase in fluorescence in the gas phase. However, two other outcomes parallel behaviour. First, the similarity of action spectra of the gaseous dsDNA-H33258 complex and the unbound diprotomer suggest that the dye may be diprotomeric when in complex with gaseous dsDNA. Second, the photodissociation power dependence measurements indicate the presence of at least two distinct populations of both H33258 in complex with dsDNA and in its unbound diprotomeric form. Overall, the results reported here reveal unexplored aspects of the potential energy landscape of H33258, including a new, stable, highly-fluorescent form that may be useful to consider in sensing applications. Moreover, the results reinforce how structure, deactivation pathways and other photophysical properties are intertwined for this DNA-binding dye, which may offer strategies for improved control of DNA-targeting drugs and sensors.
Assuntos
Bisbenzimidazol/química , DNA/química , Cor , FluorescênciaRESUMO
Detection of small amounts of RNA in various biological samples is an important applied task. Using fluorescence spectroscopy, the hydrolysis by binase of rRNA and tRNA, stained with Hoechst 33258, in aqueous solutions was investigated. The binding constant of Hoechst with rRNA is 106â¯M-1. Specific hydrolysis of rRNA and tRNA by binase during 1-2â¯min at room temperature leads to a multiple decrease in fluorescence of the dye. This rapid hydrolysis goes to large polynucleotide fragments, but not to short oligonucleotides. The binding constant of binase with rRNA is about of 2.5â¯×â¯106â¯M-1, which is several dozen times higher than with oligonucleotides. The susceptibility to binase attack depends on the secondary structure of RNA, determined by non-canonical ribonucleotides. The developed highly sensitive fluorescent method can be used for the rapid selective detection of trace amounts of rRNA or tRNA, as well as for studying the physicochemical properties of these RNAs. Using the proposed method, one can confidently detect RNA from 10-7â¯M.
Assuntos
Bisbenzimidazol/química , Endorribonucleases/química , Corantes Fluorescentes/química , RNA/análise , Limite de Detecção , Espectrometria de FluorescênciaRESUMO
The DNA hydrolysis by deoxyribonuclease (DNAse I) in aqueous solution was studied, using fluorescence spectroscopy and high-sensitive light-scattering detection. Specific hydrolysis of high-polymer DNA or fragmented DNA by the enzyme led to a strong decrease in the fluorescence of the Hoechst dye. The hydrolysis of mitochondrial DNA was accompanied by a decrease in the fluorescence of the dye only in 1.6 times. Hydrolysis within minutes and even hours led to appearance of large polynucleotide fragments, but not to short oligonucleotides, that was confirmed using polarized fluorescence and highly sensitive measurement of light-scattering. At the moment of the time of formation of a complex between DNA and DNAse I, a strong light-scattering occurred, which then dropped sharply during hydrolysis of high-molecular DNA, and slowly decreased during hydrolysis of fragmented DNA. The proposed methods can be applied for selective detection of trace amounts of various types of DNA, as well as for studying their physic-chemical properties.
Assuntos
Técnicas Biossensoriais/métodos , Bisbenzimidazol/química , DNA/análise , Desoxirribonuclease I/metabolismo , Luz , Limite de Detecção , Espalhamento de Radiação , Animais , Bovinos , DNA/química , Espectrometria de FluorescênciaRESUMO
Anion transporters have attracted substantial interest due to their ability to induce cell apoptosis by disrupting cellular anion homeostasis. In this paper we describe the synthesis, anion recognition, transmembrane anion transport and cell apoptosis-inducing activity of a series of fluorinated 1,3-bis(benzimidazol-2-yl)benzene derivatives. These compounds were synthesized from the condensation of 1,3-benzenedialdehyde or 5-fluoro-1,3-benzenedialdehyde with the corresponding 1,2-benzenediamines and fully characterized. They are able to form stable complexes with chloride anions, and exhibit potent liposomal and in vitro anionophoric activity. Their anion transport efficiency may be ameliorated by the total number of fluorine atoms, and the enhanced anionophoric activity was a likely consequence of the increased lipophilicity induced by fluorination. Most of these fluorinated bisbenzimidazoles exhibit potent cytotoxicity toward the selected cancer cells. Mechanistic investigations suggest that these compounds are able to trigger cell apoptosis probably by disrupting the homeostasis of chloride anions.
Assuntos
Apoptose/efeitos dos fármacos , Bisbenzimidazol/química , Bisbenzimidazol/farmacologia , Cloretos/química , Desenho de Fármacos , Halogenação , Linhagem Celular Tumoral , Humanos , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Relação Estrutura-AtividadeRESUMO
The interaction of atorvastatin with calf thymus DNA (CT-DNA) was investigated in vitro under simulated physiological conditions by using absorption and emission spectroscopy, viscosity measurements, gel electrophoresis and molecular docking studies. Analysis of UV-vis absorbance spectra indicates the formation of complex between atorvastatin and CT-DNA, and obtained binding constant (Kb = 8.2×104 L. mol-1) is comparable to groove binder drugs. Slight increase of viscosity of CT-DNA demonstrated the groove binding mode. Hoechst 33,258 and methylene blue (MB) displacement studies further confirmed such mode of atorvastatin interaction. Thermodynamic parameters ΔG, ΔH, and ΔS measurements were taken at different temperatures indicated that hydrophobic forces played main role in the binding process. Molecular docking provided detailed computational interaction of atorvastatin with CT-DNA which proved that atorvastatin binds to the groove of CT-DNA. Cleavage experiments showed that atorvastatin does not induce any cleavage under the experimental setup. Finally, all results indicated that atorvastatin interacts with CT-DNA via groove binding mode.
Assuntos
Atorvastatina/química , DNA/química , Simulação de Acoplamento Molecular , Ligação Competitiva , Bisbenzimidazol/química , Eletroforese em Gel Bidimensional , Azul de Metileno/química , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , ViscosidadeRESUMO
Given the increased interest in public hygiene due to outbreaks of food poisoning, increased emphasis has been placed on developing novel monitoring systems for point-of-care testing (POCT) to evaluate pathogens causing foodborne illnesses. Here, we demonstrate a pathogen evaluation system utilizing simple film-based microfluidics, featuring simultaneous gene amplification, solution mixing, and electrochemical detection. To minimize and integrate the various functionalities into a single chip, patterned polyimide and polyester films were mainly used on a polycarbonate housing chip, allowing simple fabrication and alignment, in contrast to conventional polymerase chain reaction, which requires a complex biosensing system at a bench-top scale. The individual integrated sensing chip could be manually fabricated in 10â¯min. Using the developed film-based integrated biosensing chip, the genes from the pathogens causing foodborne illnesses were simultaneously amplified based on multiple designed microfluidic chambers and Hoechst 33258, which intercalates into double-stranded DNA, to generate the electrochemical signal. The target pathogen gene was accurately analyzed by square wave voltammetry (SWV) within the 25â¯s, while the gel electrophoresis required about 30â¯min. Based on the developed integrated biosensing chip, the 1.0â¯×â¯101 and 1.0â¯×â¯102â¯colony-forming unit (CFU) of Staphylococcus aureus and Escherichia coli were sensitively detected with high reproducibility in the 25â¯s. On the basis of the significant features of the film-based molecular analysis platform, we expect that the developed sensor could be applied to the screening of various pathogens as a POCT device.
Assuntos
Técnicas Biossensoriais/métodos , Escherichia coli/isolamento & purificação , Doenças Transmitidas por Alimentos/diagnóstico , Dispositivos Lab-On-A-Chip , Intoxicação Alimentar por Salmonella/diagnóstico , Salmonella enteritidis/isolamento & purificação , Intoxicação Alimentar Estafilocócica/diagnóstico , Staphylococcus aureus/isolamento & purificação , Bisbenzimidazol/química , DNA/química , Técnicas Eletroquímicas/métodos , Escherichia coli/química , Escherichia coli/genética , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Técnicas de Amplificação de Ácido Nucleico , Testes Imediatos , Reprodutibilidade dos Testes , Salmonella enteritidis/química , Salmonella enteritidis/genética , Staphylococcus aureus/química , Staphylococcus aureus/genética , Fatores de TempoRESUMO
Optimal imaging methods are necessary in order to perform a detailed characterization of thick tissue samples from either native or engineered tissues. Tissue-engineered substitutes are featuring increasing complexity including multiple cell types and capillary-like networks. Therefore, technical approaches allowing the visualization of the inner structural organization and cellular composition of tissues are needed. This chapter describes an optical clearing technique which facilitates the detailed characterization of whole-mount samples from skin and adipose tissues (ex vivo tissues and in vitro tissue-engineered substitutes) when combined with spectral confocal microscopy and quantitative analysis on image renderings.
Assuntos
Microscopia Confocal/métodos , Imagem Óptica/métodos , Pele Artificial , Engenharia Tecidual , Tecido Adiposo/química , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/imunologia , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/imunologia , Benzotiazóis , Bisbenzimidazol/química , Bisbenzimidazol/farmacologia , Morte Celular/imunologia , Diaminas , Corantes Fluorescentes/química , Corantes Fluorescentes/farmacologia , Humanos , Compostos Orgânicos/química , Compostos Orgânicos/farmacologia , Quinolinas , Salicilatos/química , Salicilatos/farmacologia , Pele/química , Pele/efeitos dos fármacos , Pele/imunologiaRESUMO
Molecular oxygen in living cells is distributed and consumed inhomogeneously, depending on the activity of each organelle. Therefore, tractable methods that can be used to monitor the oxygen status in each organelle are needed to understand cellular function. Here we report the design of a new oxygen-sensing probe for use in the cell nucleus. We prepared "Ru-Hoechsts", each consisting of a phosphorescent ruthenium complex linked to a Hoechst 33258 moiety, and characterized their properties as oxygen sensors. The Hoechst unit shows strong DNA-binding properties in the nucleus, and the ruthenium complex shows oxygen-dependent phosphorescence. Thus, Ru-Hoechsts accumulated in the cell nucleus and showed oxygen-dependent signals that could be monitored. Of the Ru-Hoechsts prepared in this study, Ru-Hoechst b, in which the ruthenium complex and the Hoechst unit were linked through a hexyl chain, showed the most suitable properties for monitoring the oxygen status. Ru-Hoechsts are probes with high potential for visualizing oxygen fluctuations in the nucleus.