RESUMEN
We investigate the new simultaneous absorbance-transmission and fluorescence excitation-emission matrix method for rapid and effective characterization of the varying components from a mixture. The absorbance-transmission and fluorescence excitation-emission matrix method uniquely facilitates correction of fluorescence inner-filter effects to yield quantitative fluorescence spectral information that is largely independent of component concentration. This is significant because it allows one to effectively monitor quantitative component changes using multivariate methods and to generate and evaluate spectral libraries. We present the use of this novel instrument in different fields: i.e. tracking changes in complex mixtures including natural water, wine as well as monitoring stability and aggregation of hormones for biotherapeutics.
Asunto(s)
Espectrometría de Fluorescencia/métodos , Humanos , Concentración de Iones de Hidrógeno , Insulina/química , Temperatura , Agua/análisis , Vino/análisisRESUMEN
Interleukin 6 (IL6), an inflammatory response protein has major implications in immune-related inflammatory diseases. Identification of aptamers for the IL6 protein aids in diagnostic, therapeutic, and theranostic applications. Three different DNA aptamers and their interactions with IL6 protein were extensively investigated in a phosphate buffed saline (PBS) solution. Molecular-level modeling through molecular dynamics provided insights of structural, conformational changes and specific binding domains of these protein-aptamer complexes. Multiple simulations reveal consistent binding region for all protein-aptamer complexes. Conformational changes coupled with quantitative analysis of center of mass (COM) distance, radius of gyration (Rg), and number of intermolecular hydrogen bonds in each IL6 protein-aptamer complex was used to determine their binding performance strength and obtain molecular configurations with strong binding. A similarity comparison of the molecular configurations with strong binding from molecular-level modeling concurred with Surface Plasmon Resonance imaging (SPRi) for these three aptamer complexes, thus corroborating molecular modeling analysis findings. Insights from the natural progression of IL6 protein-aptamer binding modeled in this work has identified key features such as the orientation and location of the aptamer in the binding event. These key features are not readily feasible from wet lab experiments and impact the efficacy of the aptamers in diagnostic and theranostic applications.
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Aptámeros de Nucleótidos/química , Interleucina-6/química , Simulación de Dinámica Molecular , Resonancia por Plasmón de Superficie/métodos , Aptámeros de Nucleótidos/metabolismo , Enlace de Hidrógeno , Interleucina-6/metabolismo , Cinética , Conformación de Ácido Nucleico , Unión Proteica , Conformación ProteicaRESUMEN
The clinical assessment of multiple organ dysfunctions at early stages is recognized to be an important factor in prompting definitive treatment decisions that prevent irreversible organ damage. In this article, we propose a real-time, label-free, and multiplex nanoenhanced SPRi platform to quantitatively assess two biomarkers, kidney injury molecule (KIM-1) and high mobility group box-1 (HMGB-1) simultaneously in buffer. Our work involves three major contributions in the design of the immunosensor: (1) we applied site-specific immobilization of antibodies to the solid surface that avoids loss of biological activity caused by covalent attachment; (2) we constructed a well-blocked sensor surface that exhibits minimal non-specific adsorption for singleplex measurements of each biomarker in buffer; and (3) we adopted a sandwich assay that implements functionalized quantum dots (NanoEnhancers) as signal amplifiers to achieve a sensitivity level of 5 pg/mL for KIM-1 and HMGB-1 in buffer. We foresee great potential and success in extending this multiplex and ultra-sensitive platform to assess a variety of other emerging clinical biomarkers at low concentrations and in complex matrices.
Asunto(s)
Técnicas Biosensibles/métodos , Proteína HMGB1/metabolismo , Receptor Celular 1 del Virus de la Hepatitis A/metabolismo , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/metabolismo , Anticuerpos/metabolismo , Biomarcadores/metabolismo , Humanos , Fallo Hepático Agudo/diagnóstico , Fallo Hepático Agudo/metabolismo , Análisis por Matrices de Proteínas , Puntos CuánticosRESUMEN
Progesterone is a steroid hormone that plays a central role in the female reproductive processes such as ovulation and pregnancy with possible effects on other organs as well. The measurement of progesterone levels in bodily fluids can assist in early pregnancy diagnosis and can provide insight for other reproductive functions. In this work, the detection of progesterone was examined by integrating novel aptamer development with a nanoEnhanced surface plasmon resonance imaging sensor. First, we developed X-aptamers and selected them for binding to progesterone. Then, we took advantage of the multi-array feature of SPRi to develop an optimized biosensor capable of simultaneously screening the 9 X-aptamers developed to determine the binding capabilities of each aptamer. The sensor surface design conditions were further optimized for the sandwich assay, which employed nanoEnhancers (NIR-streptavidin coated quantum dots) for ultrasensitive detection of progesterone molecules. The assay designed was examined over a concentration range of 1.575 ng/mL to 126 µg/mL resulting in a limit of detection (LOD) of 1.575 ng/mL (5 nM) in phosphate buffer.
Asunto(s)
Aptámeros de Nucleótidos/química , Técnicas Biosensibles/métodos , Progesterona/análisis , Tampones (Química) , Sensibilidad y EspecificidadRESUMEN
Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml(-) (1) and minimum levels of 0.03 ng ml(-) (1)) in circulating blood are essential as variable levels may indicate altered physiology. For example, growth disorders occurring in childhood can be diagnosed by measuring levels of hGH in blood. Also, the misuse of recombinant hGH in sports not only poses an ethical issue it also presents serious health threats to the abuser. One popular strategy for measuring hGH misuse, relies on the detection of the ratio of 22 kDa hGH to total hGH, as non-22 kDa endogenous levels drop after exogenous recombinant hGH (rhGH) administration. Surface plasmon resonance imaging (SPRi) is an analytical tool that allows direct (label-free) monitoring and visualization of biomolecular interactions by recording changes of the refractive index adjacent to the sensor surface in real time. In contrast, the most frequently used colorimetric method, enzyme-linked immunosorbent assay (ELISA) uses enzyme labeled detection antibodies to indirectly measure analyte concentration after the addition of a substrate that induces a color change. To increase detection sensitivity, amplified SPRi uses a sandwich assay format and near infrared quantum dots (QDs) to increase signal strength. After direct SPRi detection of recombinant rhGH in spiked human serum, the SPRi signal is amplified by the sequential injection of detection antibody coated with near-infrared QDs (Nano-SPRi). In this study, the diagnostic potential of direct and amplified SPRi was assessed for measuring rhGH spiked in human serum and compared directly with the capabilities of a commercially available ELISA kit.
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Ensayo de Inmunoadsorción Enzimática/métodos , Hormona de Crecimiento Humana/sangre , Resonancia por Plasmón de Superficie/métodos , Humanos , Nanotecnología/métodos , Puntos Cuánticos , Proteínas Recombinantes/sangreRESUMEN
Surface plasmon resonance (SPR) is a popular technique that allows for sensitive, specific, label-free and real-time assessment of biomolecular interactions. SPR is a nondestructive, modular and flexible tool for various applications in biomedical sciences ranging from cell sorting, cell surface characterization and drug discovery. In this review, we will discuss more specifically how SPR is used to monitor the dynamics of various types of cellular binding events and morphological adherence changes in response to external stimuli.
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Rastreo Celular , Sistemas de Liberación de Medicamentos , Imagen Molecular , Resonancia por Plasmón de Superficie , Animales , Biopelículas/crecimiento & desarrollo , Fenómenos Biofísicos , Escherichia coli/crecimiento & desarrollo , Células HEK293 , HumanosRESUMEN
The fabrication of 4-mercaptobenzoic acid (4-MBA) antibody-functionalized gold nanoparticles via microwave technology for surface-enhanced Raman scattering (SERS)-based cellular nanosensing is reported. Nanoprobes were characterized by UV-vis absorbance, Raman scattering properties, and observed by TEM imaging. Results showed that microwave irradiation rapidly yielded nanoprobes with significant Raman scattering intensity and suitable stability to support antibody conjugation in under 10min. Functionalized nanoprobes demonstrated the ability to map the expression of vascular adhesion molecule-1 (VCAM-1) in human coronary artery endothelial (HCAE) cells, indicating that microwave fabrication presents a viable and rapid approach to SERS nanoprobe construction. The successful application of SERS nanoprobes to localize biomarker expression in vitro may ultimately be used for early diagnostic and preventative functions in medicine.
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Técnicas Biosensibles , Microondas , Nanoestructuras , Espectrometría Raman/métodos , Línea Celular , Humanos , Microscopía Electrónica de Transmisión , Espectrofotometría Ultravioleta , Propiedades de Superficie , Molécula 1 de Adhesión Celular Vascular/análisisRESUMEN
Diagnostic biomarkers (i.e. proteins) are often in low abundance in bodily fluids presenting many challenges for their detection. In order to extend the application of SPRi systems in detecting biomarkers at ultralow levels, we combine the advantage of aptamer technology with nanomaterials and microwave-assisted surface functionalization. By implementing a sandwich assay through the introduction of aptamer-modified quantum dots (QDs), it was possible to measure 7 zeptomole (at 5â fg/mL) of C-reactive protein (CRP) selectively in spiked human serum. It is expected that the proposed platform will provide new direction in designing ultrasensitive SPRi biosensors with multiplexing capabilities.
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Aptámeros de Nucleótidos/química , Técnicas Biosensibles/instrumentación , Proteína C-Reactiva/análisis , Nanopartículas del Metal/química , Puntos Cuánticos , Resonancia por Plasmón de Superficie/instrumentación , Aptámeros de Nucleótidos/genética , Análisis Químico de la Sangre/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Oro/química , Nanopartículas del Metal/ultraestructura , Microquímica/instrumentación , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
BACKGROUND: The hallmark of atherosclerosis is the accumulation of plaque in vessel walls. This process is initiated when monocytic cells differentiate into macrophage foam cells under conditions with high levels of atherogenic lipoproteins. Vulnerable plaque can dislodge, enter the blood stream, and result in acute myocardial infarction and stroke. Imaging techniques such as cardiovascular magnetic resonance (CMR) provides one strategy to identify patients with plaque accumulation. METHODS: We synthesized an atherosclerotic-targeting contrast agent (ATCA) in which gadolinium (Gd)-containing endohedrals were functionalized and formulated into liposomes with CD36 ligands intercalated into the lipid bilayer. In vitro assays were used to assess the specificity of the ATCA for foam cells. The ability of ATCA to detect atherosclerotic plaque lesions in vivo was assessed using CMR. RESULTS: The ATCA was able to detect scavenger receptor (CD36)-expressing foam cells in vitro and were specifically internalized via the CD36 receptor as determined by focused ion beam/scanning electron microscopy (FIB-SEM) and Western blotting analysis of CD36 receptor-specific signaling pathways. The ATCA exhibited time-dependent accumulation in atherosclerotic plaque lesions of ApoE -/- mice as determined using CMR. No ATCA accumulation was observed in vessels of wild type (C57/b6) controls. Non-targeted control compounds, without the plaque-targeting moieties, were not taken up by foam cells in vitro and did not bind plaque in vivo. Importantly, the ATCA injection was well tolerated, did not demonstrate toxicity in vitro or in vivo, and no accumulation was observed in the major organs. CONCLUSIONS: The ATCA is specifically internalized by CD36 receptors on atherosclerotic plaque providing enhanced visualization of lesions under physiological conditions. These ATCA may provide new tools for physicians to non-invasively detect atherosclerotic disease.
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Aorta/patología , Enfermedades de la Aorta/diagnóstico , Aterosclerosis/diagnóstico , Medios de Contraste , Fulerenos , Imagen por Resonancia Magnética , Compuestos Organometálicos , Placa Aterosclerótica , Animales , Aorta/metabolismo , Enfermedades de la Aorta/metabolismo , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Antígenos CD36/metabolismo , Medios de Contraste/metabolismo , Medios de Contraste/toxicidad , Modelos Animales de Enfermedad , Células Espumosas/metabolismo , Células Espumosas/patología , Fulerenos/metabolismo , Fulerenos/toxicidad , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Rastreo , Compuestos Organometálicos/metabolismo , Compuestos Organometálicos/toxicidad , Valor Predictivo de las Pruebas , Factores de Tiempo , Células U937RESUMEN
AIM: To enhance the stability of siRNA while improving their therapeutic properties and visualization at the target site, a novel nanoplex system was developed. MATERIALS & METHODS: The designed nanoplex system involved functionalizing siRNA with near-infrared quantum dots and loading them into histidylated glycol chitosan (GC-His). RESULTS: Colocalization studies revealed a twofold increase in siRNA uptake after encapsulation with GC-His and nanoparticles were localized in cytoplasm, suggesting that histidine promoted their dissociation from the endosomal membranes. Furthermore, as opposed to siRNAs treated with commercial transfection reagent, siRNAs loaded within GC-His showed a marked reduction (64%) of MDM2 protein expression 24 h after transfection. CONCLUSION: These findings concur that GC-His/siRNA-quantum dot nanoplexes are promising multifunctional vehicles for gene inhibitory therapy.
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Terapia Genética/métodos , Nanopartículas/química , Western Blotting , Línea Celular Tumoral , Quitosano/química , Ensayo de Cambio de Movilidad Electroforética , Humanos , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Estructura Molecular , Proteínas Proto-Oncogénicas c-mdm2/genética , Proteínas Proto-Oncogénicas c-mdm2/metabolismo , Puntos CuánticosRESUMEN
The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 µg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QD's mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.
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Técnicas Biosensibles , Puntos Cuánticos , Espectroscopía Infrarroja Corta/métodos , Resonancia por Plasmón de Superficie/métodos , Secuencia de Bases , Cartilla de ADN , ADN de Cadena Simple/análisis , Límite de Detección , Antígeno Prostático Específico/análisisRESUMEN
AIM: To assess the effects of oleic acid treatment on subcellular distribution of indium gallium phosphide-zinc sulfide (InGaP/ZnS) nanoparticles in microglia and astrocytes. MATERIALS & METHODS: The extent of colocalization between the nanoparticles and organelles was assessed by confocal microscopy, spectrofluorometry and cell sorting. RESULTS: Cell treatment with a common fatty acid (oleic acid) within the range of physiological concentrations markedly enhanced the InGaP/ZnS uptake by microglia and afforded their colocalization within lipid droplets/lysosomes but not with mitochondria. CONCLUSION: These results suggest that the availability of mono-unsaturated fatty acids, such as oleic acid, in different cells could significantly alter nanoparticle uptake and localization, which can in turn affect the functions of cells and tissues coexposed to nanoparticles.
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Galio/metabolismo , Indio/metabolismo , Nanopartículas/química , Nanotecnología/métodos , Neuroglía/metabolismo , Fosfinas/metabolismo , Sulfuros/metabolismo , Compuestos de Zinc/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Galio/química , Indio/química , Ratones , Microscopía Confocal , Neuroglía/efectos de los fármacos , Ácido Oléico/farmacología , Proteínas Asociadas a Pancreatitis , Fosfinas/química , Sulfuros/química , Compuestos de Zinc/químicaRESUMEN
Prodrugs are biologically inactive derivatives of an active drug intended to solve certain problems of the parent drug such as toxicity, instability, minimal solubility and non-targeting capabilities. The majority of drugs for cardiovascular diseases undergo first-pass metabolism, resulting in drug inactivation and generation of toxic metabolites, which makes them appealing targets for prodrug design. Since prodrugs undergo a chemical reaction to form the parent drug once inside the body, this makes them very effective in controlling the release of a variety of compounds to the targeted site. This review will provide the reader with an insight on the latest developments of prodrugs that are available for treating a variety of cardiovascular diseases. In addition, we will focus on several drug delivery methodologies that have merged with the prodrug approach to provide enhanced target specificity and controlled drug release with minimal side effects.
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Enfermedades Cardiovasculares/tratamiento farmacológico , Profármacos/uso terapéutico , Sistemas de Liberación de Medicamentos , Humanos , Profármacos/administración & dosificación , Profármacos/químicaAsunto(s)
Metalotioneína/fisiología , Nanopartículas/química , Nanotecnología/instrumentación , Nanotecnología/métodos , Semiconductores , Compuestos de Cadmio , Espectroscopía de Resonancia por Spin del Electrón , Radicales Libres , Espectrometría de Masas/métodos , Metalotioneína/química , Nanoestructuras/química , Óxido Nítrico/química , Péptidos/química , Proteínas/química , Especies Reactivas de Oxígeno , Compuestos de Selenio , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Sulfuros/química , Compuestos de Zinc/químicaRESUMEN
Reagentless and reversible maltose biosensors are demonstrated using ZnS coated CdSe (CdSe@ZnS) nanoparticle emission intensities. This method is based on electron transfer quenching of unimolecular protein-CdSe@ZnS nanoparticle assemblies, which is provided by a protein-attached Ru(II) complex. This Ru(II) complex is presumed to reduce a valence band hole of the CdSe@ZnS excited state by tunneling through the ZnS overcoating. The Ru(II) complex mediated quenching of CdSe@ZnS nanoparticle emission was only decreased 1.2-fold relative to the CdSe nanoparticle systems. While four different Ru(II) complex attachment sites provided different amounts of nanoparticle emission quenching (1.20 to 1.75-fold decrease), all of these attachment sites yielded maltose-dependent intensity changes (1.1 to 1.4-fold increase upon maltose addition). Maltose dissociation constants for these four biosensing systems range from 250 nM to 1.0 microM, which are similar to the maltose-maltose binding protein dissociation constant that these sensors are based on. The increased fluorescence intensity was found to only occur in the presence of maltose. Furthermore, the ability of these reagentless protein-nanoparticle assemblies to perform maltose biosensing reversibly is demonstrated with the addition of alpha-glucosidase. Three 50 microM maltose additions after alpha-glucosidase addition showed increases of 2.2 microM, 600 nM, and 150 nM maltose. This result demonstrates a fluorometric method for examining alpha-glucosidase activity. Using maltose binding protein to control Ru(II) complex interactions with CdSe@ZnS nanoparticle surfaces provide a novel class of highly fluorescent, photostable biosensors that are selective for maltose.
Asunto(s)
Técnicas Biosensibles/métodos , Maltosa/análisis , Técnicas Biosensibles/instrumentación , Proteínas Portadoras , Proteínas de Unión a Maltosa , Nanotecnología , Nanotubos , Semiconductores , Espectrometría de Fluorescencia/métodosRESUMEN
Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.
Asunto(s)
Técnicas Biosensibles , Nanoestructuras/química , Compuestos de Cadmio/química , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Maltosa/química , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Tamaño de la Partícula , Reproducibilidad de los Resultados , Compuestos de Selenio/química , Espectrometría de FluorescenciaRESUMEN
Metallothionein fusion proteins allow for site-specific, orthogonal functionalization of proteins to a variety of nanoparticles.