RESUMO
A novel AIEgen molecular probe (N-3QL) with typical AIE effects, good biocompatibility, lysosome targeting, pH activation, excellent photostability, and high brightness was synthesized using two simple synthetic steps. Spectroscopic and cytotoxicity experiments indicate that N-3QL can not only be used for the dynamic monitoring of cancer cell lysosomes, but also for photodynamic therapy (PDT) ablation of cancer cells.
Assuntos
Fotoquimioterapia , Fotoquimioterapia/métodos , Sondas Moleculares/análise , Concentração de Íons de Hidrogênio , Lisossomos/químicaRESUMO
PURPOSE: Boramino acids are a class of amino acid biomimics that replace the carboxylate group with trifluoroborate and can achieve the 18F-labeled positron emission tomography (PET) and boron neutron capture therapy (BNCT) with identical chemical structure. METHODS: This study reports a trifluoroborate-derived boronophenylalanine (BBPA), a derived boronophenylalanine (BPA) for BNCT, as a promising PET tracer for tumor imaging. RESULTS: Competition inhibition assays in cancer cells suggested the cell accumulation of [18F]BBPA is through large neutral amino acid transporter type-1 (LAT-1). Of note, [18F]BBPA is a pan-cancer probe that shows notable tumor uptake in B16-F10 tumor-bearing mice. In the patients with gliomas and metastatic brain tumors, [18F]BBPA-PET shows good tumor uptake and notable tumor-to-normal brain ratio (T/N ratio, 18.7 ± 5.5, n = 11), higher than common amino acid PET tracers. The [18F]BBPA-PET quantitative parameters exhibited no difference in diverse contrast-enhanced status (P = 0.115-0.687) suggesting the [18F]BBPA uptake was independent from MRI contrast-enhancement. CONCLUSION: This study outlines a clinical trial with [18F]BBPA to achieve higher tumor-specific accumulation for PET, provides a potential technique for brain tumor diagnosis, and might facilitate the BNCT of brain tumors.
Assuntos
Compostos de Boro , Neoplasias Encefálicas , Radioisótopos de Flúor , Fenilalanina , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Traçadores Radioativos , Animais , Feminino , Humanos , Camundongos , Compostos de Boro/análise , Compostos de Boro/metabolismo , Compostos de Boro/farmacocinética , Terapia por Captura de Nêutron de Boro , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Neoplasias Encefálicas/diagnóstico por imagem , Neoplasias Encefálicas/metabolismo , Radioisótopos de Flúor/análise , Radioisótopos de Flúor/metabolismo , Radioisótopos de Flúor/farmacocinética , Voluntários Saudáveis , Transportador 1 de Aminoácidos Neutros Grandes/metabolismo , Imageamento por Ressonância Magnética , Melanoma Experimental , Camundongos Endogâmicos C57BL , Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Sondas Moleculares/farmacocinética , Fenilalanina/análogos & derivados , Fenilalanina/análise , Fenilalanina/metabolismo , Fenilalanina/farmacocinética , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Deciphering the dynamics of intracellular organelles has gained immense attention due to their subtle control over diverse, complex biological processes such as cellular metabolism, energy homeostasis, and autophagy. In this context, molecular materials, including small-organic fluorescent probes and their supramolecular self-assembled nano-/microarchitectures, have been employed to explore the diverse intracellular biological events. However, only a handful of fluorescent probes and self-assembled emissive structures have been successfully used to track different organelle's movements, circumventing the issues related to water solubility and long-term photostability. Thus, the water-soluble molecular fluorescent probes and the water-dispersible supramolecular self-assemblies have emerged as promising candidates to explore the trafficking of the organelles under diverse physiological conditions. In this review, we have delineated the recent progress of fluorescent probes and their supramolecular self-assemblies for the elucidation of the dynamics of diverse cellular organelles with a special emphasis on lysosomes, lipid droplets, and mitochondria. Recent advancement in fluorescence lifetime and super-resolution microscopy imaging has also been discussed to investigate the dynamics of organelles. In addition, the fabrication of the next-generation molecular to supramolecular self-assembled luminogens for probing the variation of microenvironments during the trafficking process has been outlined.
Assuntos
Corantes Fluorescentes , Organelas , Corantes Fluorescentes/análise , Lisossomos/metabolismo , Mitocôndrias/química , Sondas Moleculares/análise , Organelas/química , Água/análiseRESUMO
In order to prevent and control the effects of pesticide residues on human health and the ecological environment, the rapid, highly sensitive, and selective detection of multiple pesticide residues has become an urgent problem to be solved. Herein, a lab-on-a-molecule probe based on a host-guest complex (ThT@Q[8] probe) has been developed to simultaneously analyze multiple aromatic pesticides under single wavelength excitation, such as fuberidazole, thiabendazole, carbendazim, thidiazuron, and tricyclazole. The fluorescence titration spectra of the ThT@Q[8] probe with the five pesticides mentioned above showed that the fluorescence intensity exhibited a good linear correlation with the pesticide concentration and the limit of detection was as low as 10-7 M. Because the ThT@Q[8] probe exhibits diverse fluorescence color changes to the five pesticides studied under a 365 nm ultraviolet lamp, we fabricated a single probe used to detect multiple analytes in the RGB triple channel by extracting the RGB variations. Principal component analysis and linear discriminant analysis proved that the ThT@Q[8] probe can recognize and distinguish five pesticides and can be applied at different concentrations. In real samples, the ThT@Q[8] probe recognized and distinguished five pesticides in tap water and Huaxi River water. The 1H NMR spectra results proved that a charge-transfer complex of ThT and pesticides in the Q[8] cavity may be formed. Moreover, we selected a test strip as a carrier to detect pesticides. The results indicate it can be used to quickly and conveniently detect different pesticides due to the rapid color change. Besides, the ThT@Q[8] probe has good cell permeability and can be used to detect pesticide residues in living cells. This work has laid the foundation for the qualitative and quantitative multitarget detection of pesticide residues.
Assuntos
Resíduos de Praguicidas , Praguicidas , Humanos , Sondas Moleculares/análise , Resíduos de Praguicidas/análise , Praguicidas/análise , Espectrometria de Fluorescência/métodos , Água/análiseRESUMO
The cyclic signal amplification technology has been widely applied for the ultrasensitive detection of many important biomolecules, such as nucleic acids, proteins, enzymes, adenosine triphosphate (ATP), metal ions, exosome, etc. Due to their low content in the complex biological samples, traditional detection methods are insufficient to satisfy the requirements for monitoring those biomolecules. Therefore, effective and sensitive biosensors based on cyclic signal amplification technology are of great significance for the quick and simple diagnosis and treatment of diseases. Fluorescent biosensor based on cyclic signal amplification technology has become a research hotspot due to its simple operation, low cost, short time, high sensitivity and high specificity. This paper introduces several cyclic amplification methods, such as rolling circle amplification (RCA), strand displacement reactions (SDR) and enzyme-assisted amplification (EAA), and summarizes the research progress of using this technology in the detection of different biomolecules in recent years, in order to provide help for the research of more efficient and sensitive detection methods.
Assuntos
Técnicas Biossensoriais/métodos , Corantes Fluorescentes , Sondas Moleculares , Técnicas de Amplificação de Ácido Nucleico/métodos , Ácidos Nucleicos , Animais , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Humanos , Camundongos , Sondas Moleculares/análise , Sondas Moleculares/química , Ácidos Nucleicos/análise , Ácidos Nucleicos/química , Ácidos Nucleicos/metabolismo , Proteínas/análise , Proteínas/químicaRESUMO
The mammalian sirtuins are a group of posttranslational modification enzymes that remove acyl modifications from lysine residues in an NAD+-dependent manner. Although initially proposed as histone deacetylases (HDACs), they are now known to target other cellular enzymes and proteins as well. Sirtuin-catalyzed simple amide hydrolysis has profound biological consequences including suppression of gene expression, promotion of DNA damage repair, and regulation of glucose and lipid metabolism. Human sirtuins have been intensively pursued by both academia and industry as potential therapeutic targets for the treatment of diseases such as cancer and neurodegeneration. To gain a better understanding of their roles in various cellular events, innovative chemical probes are highly sought after. This current study focuses on the development of activity-based chemical probes (ABPs) for the profiling of sirtuin activity in biological samples. Cyclooctyne-containing and azido-containing probes were synthesized to enable the subsequent copper-free "click" conjugation to either a fluorophore or biotin. The two groups of structurally related ABPs demonstrated different labeling efficiency and selectivity: the cyclooctyne-containing probes failed to label recombinant sirtuins to any appreciable level, while the azido-containing ABPs showed good isoform selectivity. The azido-containing ABPs were further analyzed for their ability to label an individual sirtuin isoform in protein mixtures and cell lysates. These biocompatible ABPs allow the study of dynamic cellular protein activity change to become possible.
Assuntos
Química Click/métodos , Sirtuínas/metabolismo , Animais , Azidas/análise , Azidas/metabolismo , Ensaios Enzimáticos/métodos , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Humanos , Sondas Moleculares/análise , Sondas Moleculares/metabolismo , Sirtuínas/análiseRESUMO
The skin is exposed to various external stimuli. Keratinocytes, which are the main cell type in the epidermis, interact with peripheral sensory neurons and modulate neuronal activity. Recent studies have revealed that keratinocytes play crucial roles in nociception, and that ATP is one of the main mediators of signal transduction from keratinocytes to sensory neurons. However, no quantitative cellular level analyses of ATP-mediated information flow from keratinocytes to sensory dorsal root ganglion (DRG) neurons have been conducted. In this study, we performed simultaneous imaging of cell surface ATP and intracellular Ca2+ signals using both iATPSnFR, a genetically encoded ATP probe localized to the outside of the cell membrane, and the Ca2+ probe, Fura-red. Upon mechanical stimulation of the keratinocyte with a glass needle, an increase in Ca2+ and ATP release were observed around the stimulated area, and these phenomena were positively correlated. In cultured DRG neurons and keratinocytes neighboring the stimulated keratinocyte, increased intracellular Ca2+ concentration and levels of cell surface ATP on the side closer to the stimulated cell were detected. The ratio of Ca2+ response to input ATP signal was significantly larger in DRG neurons than in keratinocytes. We found that DRG neurons were more sensitive to ATP than keratinocytes, and therefore, only DRG neurons responded to ATP at 1 µM or lower concentrations when in co-culture with keratinocytes. Moreover, signals caused by moderate mechanical stimulation of keratinocytes were transmitted predominantly to DRG neurons. These findings would be important in the further determination of the detailed mechanism of nociception in the epidermis.
Assuntos
Trifosfato de Adenosina/farmacologia , Cálcio/metabolismo , Queratinócitos/efeitos dos fármacos , Mecanotransdução Celular , Células Receptoras Sensoriais/efeitos dos fármacos , Trifosfato de Adenosina/metabolismo , Animais , Benzofuranos/análise , Benzofuranos/química , Cátions Bivalentes , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Técnicas de Cocultura , Epiderme/inervação , Epiderme/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/metabolismo , Genes Reporter , Humanos , Imidazóis/análise , Imidazóis/química , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/metabolismo , Sondas Moleculares/análise , Sondas Moleculares/química , Nociceptividade/fisiologia , Ratos , Ratos Wistar , Células Receptoras Sensoriais/citologia , Células Receptoras Sensoriais/metabolismo , Imagem com Lapso de TempoRESUMO
Despite the importance of rapid and accurate detection of SARS-CoV-2 in controlling the COVID-19 pandemic, current diagnostic methods are static and unable to distinguish between viable/nonviable virus or directly reflect viral replication activity. Real-time imaging of protease activity specific to SARS-CoV-2 can overcome these issues but remains lacking. Herein, we report a near-infrared fluorescence (NIRF) activatable molecular probe (SARS-CyCD) for detection of SARS-CoV-2 protease in living mice. The probe comprises a hemicyanine fluorophore caged with a protease peptide substrate and a cyclodextrin unit, which function as an NIRF signaling moiety and a renal-clearable enabler, respectively. The peptide substrate of SARS-CyCD can be specifically cleaved by SARS-CoV-2 main protease (Mpro), resulting in NIRF signal activation and liberation of the renal-clearable fluorescent fragment (CyCD). Such a design not only allows sensitive detection of Mpro in the lungs of living mice after intratracheal administration but also permits optical urinalysis of SARS-CoV-2 infection. Thus, this study presents an in vivo sensor that holds potential in preclinical high-throughput drug screening and clinical diagnostics for respiratory viral infections.
Assuntos
COVID-19/diagnóstico , Rim/metabolismo , Sondas Moleculares/metabolismo , Imagem Óptica/métodos , Animais , COVID-19/virologia , Corantes Fluorescentes/análise , Corantes Fluorescentes/metabolismo , Humanos , Pulmão/metabolismo , Camundongos , Sondas Moleculares/análise , SARS-CoV-2/enzimologia , SARS-CoV-2/isolamento & purificação , Espectroscopia de Luz Próxima ao Infravermelho , Urinálise , Proteínas da Matriz Viral/metabolismoAssuntos
Fluorescência , Proteínas Luminescentes/análise , Sondas Moleculares/análise , Imagem Óptica/métodos , Animais , Biliverdina/genética , Biliverdina/metabolismo , Cor , Escherichia coli/genética , Escherichia coli/metabolismo , Citometria de Fluxo/métodos , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Raios Infravermelhos , Medições Luminescentes , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Sondas Moleculares/genética , Sondas Moleculares/metabolismo , Proteína Vermelha FluorescenteRESUMO
This protocol describes how to culture, image, and determine the nuclear position of a fluorescently tagged DNA locus in the 3D nucleoplasm of fixed Saccharomyces cerevisiae cells. Here, we propose a manual scoring method based on widefield images and an automated method based on 3D-SIM images. Yeast culture conditions have to be followed meticulously to get the best biological response in a given environment. For complete details on the use and execution of this protocol, please refer to Forey et al. (2020).
Assuntos
Núcleo Celular/química , DNA , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Saccharomyces cerevisiae , DNA/análise , DNA/química , DNA/metabolismo , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Sondas Moleculares/análise , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologiaRESUMO
Nanoparticle internalisation is crucial for the precise delivery of drug/genes to its intracellular targets. Conventional quantification strategies can provide the overall profiling of nanoparticle biodistribution, but fail to unambiguously differentiate the intracellularly bioavailable particles from those in tumour intravascular and extracellular microenvironment. Herein, we develop a binary ratiometric nanoreporter (BiRN) that can specifically convert subtle pH variations involved in the endocytic events into digitised signal output, enabling the accurately quantifying of cellular internalisation without introducing extracellular contributions. Using BiRN technology, we find only 10.7-28.2% of accumulated nanoparticles are internalised into intracellular compartments with high heterogeneity within and between different tumour types. We demonstrate the therapeutic responses of nanomedicines are successfully predicted based on intracellular nanoparticle exposure rather than the overall accumulation in tumour mass. This nonlinear optical nanotechnology offers a valuable imaging tool to evaluate the tumour targeting of new nanomedicines and stratify patients for personalised cancer therapy.
Assuntos
Antineoplásicos/administração & dosagem , Portadores de Fármacos/análise , Imagem Molecular/métodos , Nanopartículas/análise , Neoplasias/tratamento farmacológico , Animais , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Doxorrubicina/administração & dosagem , Portadores de Fármacos/administração & dosagem , Portadores de Fármacos/química , Feminino , Corantes Fluorescentes/química , Humanos , Microscopia Intravital , Camundongos , Sondas Moleculares/administração & dosagem , Sondas Moleculares/análise , Sondas Moleculares/química , Nanopartículas/administração & dosagem , Nanopartículas/química , Neoplasias/diagnóstico por imagem , Neoplasias/patologia , Imagem Óptica/métodos , Paclitaxel/administração & dosagem , Seleção de Pacientes , Reprodutibilidade dos Testes , Nanomedicina Teranóstica/métodos , Distribuição Tecidual , Microambiente Tumoral/efeitos dos fármacosRESUMO
Due to the rigid structure of 1,3,5-triaza-7-phosphaadamantane (PTA), its 31P chemical shift solely depends on non-covalent interactions in which the molecule is involved. The maximum range of change caused by the most common of these, hydrogen bonding, is only 6 ppm, because the active site is one of the PTA nitrogen atoms. In contrast, when the PTA phosphorus atom is coordinated to a metal, the range of change exceeds 100 ppm. This feature can be used to support or reject specific structural models of organometallic transition metal complexes in solution by comparing the experimental and Density Functional Theory (DFT) calculated values of this 31P chemical shift. This approach has been tested on a variety of the metals of groups 8-12 and molecular structures. General recommendations for appropriate basis sets are reported.
Assuntos
Adamantano/análogos & derivados , Complexos de Coordenação/química , Espectroscopia de Ressonância Magnética/métodos , Sondas Moleculares/análise , Compostos Organometálicos/química , Compostos Organofosforados/química , Fósforo/análise , Elementos de Transição/química , Adamantano/química , Catálise , Estrutura Molecular , ÁguaRESUMO
Quantitative analysis using a turn-on fluorescent probe is inherently difficult due to the dependency of the fluorescence intensity on the probe concentration. To overcome this limitation, we developed an in situ quantification method using a turn-on fluorescent probe and a standard fluorophore, which are colocalized by protein tag technology. This protocol describes the synthesis of a Zn2+ probe, named ZnDA-1H, and the procedure to quantify the labile Zn2+ concentration in the Golgi of live HeLa cells by confocal fluorescence microscopy. For complete details on the use and execution of this protocol, please refer to Kowada et al. (2020).
Assuntos
Corantes Fluorescentes , Complexo de Golgi , Sondas Moleculares , Zinco , Corantes Fluorescentes/análise , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Sondas Moleculares/análise , Sondas Moleculares/síntese química , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Zinco/análise , Zinco/metabolismoRESUMO
In times of widespread multiple antibiotic resistance, the bacterial colonization of crucial medical surfaces should be detected as fast as possible. In this work, we present the non-destructive SERS method for the detection of bacterial colonization. SERS is an excellent tool for the monitoring of suitable substances in low concentrations. The SERS substrate was prepared by the aggregation of citrate-stabilized gold nanoparticles and the adsorption of the reporters (crystal violet, thiamine, and adenine). We have tested the substrate for the detection of clinically relevant S. aureus and P. aeruginosa bacteria. The SERS spectra before and after the substrate incubation revealed the degradation of the reporter by the growing bacteria. The growth of P. aeruginosa was detected using the substrates with preadsorbed crystal violet or adenine. The suitable reporter for the detection of S. aureus remains to be discovered. The selection of the reporters resistant to exposure but easily degraded by bacteria will open the way for the in situ monitoring of bacterial colonization, thus complementing the arsenal of methods in the battle against hospital infections.
Assuntos
Adenina/química , Violeta Genciana/química , Pseudomonas aeruginosa/crescimento & desenvolvimento , Pseudomonas aeruginosa/isolamento & purificação , Análise Espectral Raman/métodos , Ácido Cítrico/química , Ouro/química , Nanopartículas Metálicas/análise , Nanopartículas Metálicas/química , Sondas Moleculares/análise , Sondas Moleculares/química , Staphylococcus aureus , Tiamina/químicaAssuntos
Anticorpos/uso terapêutico , Vacinas contra COVID-19 , Biologia Celular , Biologia do Desenvolvimento , Nariz Eletrônico , Espectrometria de Massas/instrumentação , Neurociências , Animais , Anticorpos/química , Anticorpos/genética , Anticorpos/imunologia , Proteínas de Bactérias/efeitos dos fármacos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/efeitos da radiação , Bioimpressão/tendências , COVID-19/epidemiologia , COVID-19/imunologia , COVID-19/prevenção & controle , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Vacinas contra COVID-19/provisão & distribuição , Biologia Celular/instrumentação , Biologia Celular/tendências , Biologia do Desenvolvimento/métodos , Biologia do Desenvolvimento/tendências , Embrião de Mamíferos/citologia , Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário/genética , Holografia/tendências , Humanos , Imunoglobulina E/química , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina E/uso terapêutico , Canais Iônicos/metabolismo , Espectrometria de Massas/métodos , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/efeitos da radiação , Camundongos , Microscopia/instrumentação , Microscopia/tendências , Sondas Moleculares/análise , Neoplasias/tratamento farmacológico , Neurociências/métodos , Neurociências/tendências , Optogenética/tendências , Análise de Célula Única , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Four-stranded G-quadruplex (G4) DNA is a non-canonical DNA topology that has been proposed to form in cells and play key roles in how the genome is read and used by the cellular machinery. Previously, a fluorescent triangulenium probe (DAOTA-M2) was used to visualise G4s in cellulo, thanks to its distinct fluorescence lifetimes when bound to different DNA topologies. Herein, the library of available triangulenium probes is expanded to explore how modifications to the fluorescent core of the molecule affect its photophysical characteristics, interaction with DNA and cellular localisation. The benzo-bridged and isopropyl-bridged diazatriangulenium dyes, BDATA-M2 and CDATA-M2 respectively, featuring ethyl-morpholino substituents, were synthesised and characterised. The interactions of these molecules with different DNA topologies were studied to determine their binding affinity, fluorescence enhancement and fluorescence lifetime response. Finally, the cellular uptake and localisation of these optical probes were investigated. Whilst structural modifications to the triangulenium core only slightly alter the binding affinity to DNA, BDATA-M2 and CDATA-M2 cannot distinguish between DNA topologies through their fluorescence lifetime. It is argued theoretically and experimentally that this is due to reduced effectiveness of photoinduced electron transfer (PET) quenching. This work presents valuable new evidence into the critical role of PET quenching when using the fluorescence lifetime of triangulenium dyes to discriminate G4 DNA from duplex DNA, highlighting the importance of fine tuning redox and spectral properties when developing new triangulenium-based G4 probes.
Assuntos
DNA/análise , DNA/química , Fluorescência , Corantes Fluorescentes/química , Quadruplex G , Transporte de Elétrons , Corantes Fluorescentes/análise , Sondas Moleculares/análise , Sondas Moleculares/químicaRESUMO
The influence of experimental conditions on chromatographic behaviour of promising oligodeoxynucleotide double-labelled molecular probes containing an azaphthalocyanine macrocycle as a perspective dark quencher was studied. A recently introduced new stationary phase based on styrene-divinylbenzene copolymer was tested. The planar and hydrophobic structure of the azaphthalocyanine is considerably different from those of currently used fluorophores and quenchers. Thus, the most challenging issue was the separation of the double-labelled probe from its main impurity represented by a mono-labelled probe, containing only the azaphthalocyanine macrocycle. The absorbance measurement cannot simply determine this impurity, and its presence fundamentally compromises the biological assay. The commonly used gradient elution was not suitable and isocratic conditions seemed to be more appropriate. The azaphthalocyanine moiety influences the properties of the modified oligodeoxynucleotides substantially, and thus their chromatographic behaviour was determined predominantly by this quencher. Acetonitrile was the preferred organic solvent for the analysis of probes containing the azaphthalocyanine quencher and the effect of ion-pairing reagents was dependent on the probe structure. The temperature seemed to be an effective parameter for fine-tuning of the separation and mass transfer improvement. Generally, our findings could be helpful in method development for purity evaluation of double-labelled oligodeoxynucleotide probes and semipreparative methods.
Assuntos
Compostos Aza , Cromatografia Líquida de Alta Pressão/métodos , Corantes Fluorescentes , Sondas Moleculares , Oligodesoxirribonucleotídeos , Acetonitrilas/química , Compostos Aza/análise , Compostos Aza/química , Corantes Fluorescentes/análise , Corantes Fluorescentes/química , Interações Hidrofóbicas e Hidrofílicas , Sondas Moleculares/análise , Sondas Moleculares/química , Oligodesoxirribonucleotídeos/análise , Oligodesoxirribonucleotídeos/química , SolventesRESUMO
DNA-based probes constitute a versatile platform for making biological measurements due to their ability to recognize both nucleic acid and non-nucleic acid targets, ease of synthesis and chemical modification, amenability to be interfaced with signal amplification schemes, and inherent biocompatibility. Here, we provide a historical perspective of how a transition from linear DNA structures toward more structurally complex nanostructures has revolutionized live-cell analysis. Modulating the structure gives rise to probes that can enter cells without the aid of transfection reagents and can detect, track, and quantify analytes in live cells at the single-organelle, single-cell, tissue section, and whole organism levels. We delineate the advantages and disadvantages associated with different probe architectures and describe the advances enabled by these structures for elucidating fundamental biology as well as developing improved diagnostic and theranostic systems. We also discuss the outstanding challenges in the field and outline potential solutions.
Assuntos
Sobrevivência Celular , Células , DNA/química , Sondas Moleculares/análise , Sondas Moleculares/química , Nanoestruturas/química , Animais , Transferência Ressonante de Energia de Fluorescência , Humanos , Neoplasias/diagnóstico por imagem , Neoplasias Experimentais/diagnóstico por imagemRESUMO
A FRET-based probe for mapping the fluctuation of ONOO- in cisplatin-induced acute kidney injury was constructed. It exhibits ratiometric near infrared fluorescence and a dramatic decrease of its peak absorbance at 719 nm upon addition of ONOO- that is converted into remarkable signal changes in fluorescence and photoacoustic images respectively.