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1.
Adv Exp Med Biol ; 1148: 105-114, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31482496

RESUMO

Fluorescence spectroscopy is one of the most important techniques in the study of therapeutic enzymes. The fluorescence phenomenon has been discovered and exploited for centuries, while therapeutic enzymes have been used in treatment of disease for only decades. This chapter provides a brief summary of the current applications of fluorescence methods in studying therapeutic enzymes to provide some insights on the selection of proper method tailored to the goal. First a brief introduction about therapeutic enzymes and history of fluorescence were provided, followed by discussions on how fluorescence was applied in the studies. Four popular fluorescence methods are discussed: fluorescence tracing, fluorescence resonance energy transfer (FRET), fluorescence quenching and fluorescence polarization. Selected application of the fluorescence methods in studying therapeutic enzymes are listed, and discussed in details in the following paragraphs.


Assuntos
Enzimas/química , Transferência Ressonante de Energia de Fluorescência , Espectrometria de Fluorescência , Enzimas/farmacologia , Coloração e Rotulagem
2.
Chem Commun (Camb) ; 55(76): 11442-11445, 2019 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-31486469

RESUMO

Plasmonic resonance energy transfer (PRET) from a Au nanosphere (AuNS) to a quantum dot (QD) is discovered at the single particle level. A homogenous immunoassay based on this PRET is verified using a prostate specific antigen (PSA) as an example. The limit of detection of the PSA is determined to be 0.2 fM.


Assuntos
Técnicas Biossensoriais , Transferência Ressonante de Energia de Fluorescência , Ouro/química , Imunoensaio , Nanopartículas Metálicas/química , Pontos Quânticos/química , Albuminas/análise , Aminoácidos/sangue , Animais , Ânions/sangue , Biomarcadores/sangue , Cátions/sangue , Galinhas , Humanos
3.
Nat Methods ; 16(9): 815-829, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31471616

RESUMO

The applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.


Assuntos
Pesquisa Biomédica , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Animais , Humanos
4.
Chem Commun (Camb) ; 55(80): 12052-12055, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31535680

RESUMO

In this paper we report the kinetics based detection of single nucleotide variation (SNV) at room temperature by allele specific extension with different concentrations and types of crowding agents. In general, the crowding conditions enhanced the specificity in the detection of SNV.


Assuntos
DNA/genética , Nucleotídeos/genética , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Pareamento Incorreto de Bases , Técnicas Biossensoriais/métodos , Bovinos , Dextranos/química , Transferência Ressonante de Energia de Fluorescência/métodos , Corantes Fluorescentes/química , Cinética , Polietilenoglicóis/química , Soroalbumina Bovina/química , Temperatura Ambiente
5.
Chem Commun (Camb) ; 55(66): 9829-9832, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31363730

RESUMO

Polyethylene glycol grafted pyrrole-based conjugated polymers are synthesized through a one-pot multicomponent methodology, the self-assemblies of which enable nanoparticle size-selective encapsulation of drug molecules and their sustained release. Efficient loading of curcumin through drug-nanoparticle core interactions is probed using FRET, and the inherently fluorescent nature of polypyrrole could be used to detect these nanocarriers intracellularly.


Assuntos
Portadores de Fármacos , Nanopartículas/química , Polietilenoglicóis/química , Polímeros/química , Pirróis/química , Linhagem Celular Tumoral , Curcumina/administração & dosagem , Transferência Ressonante de Energia de Fluorescência , Humanos , Interações Hidrofóbicas e Hidrofílicas , Concentração Inibidora 50 , Microscopia Eletrônica de Transmissão , Espectrofotometria Ultravioleta
6.
Chem Commun (Camb) ; 55(66): 9857-9860, 2019 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-31364637

RESUMO

A novel fluorescent sensing platform for telomerase activity assay was developed by coupling a three-dimensional (3D) DNA walker with the MnO2 nanosheet-upconversion nanoparticle (UCNPs) complex-based fluorescence resonance energy-transfer (FRET) system.


Assuntos
Técnicas Biossensoriais/métodos , DNA/química , Transferência Ressonante de Energia de Fluorescência/instrumentação , Compostos de Manganês/química , Nanoestruturas/química , Óxidos/química , Telomerase/metabolismo , Limite de Detecção
7.
Chem Commun (Camb) ; 55(72): 10776-10779, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31432809

RESUMO

Herein, we present a dual-site ratiometric fluorescent probe based on the ICT-PET-FRET mechanism for the detection of pH in the ER. The probe showed a highly sensitive response to pH in the range of 5.0-7.2, and could be applied for the quantitative measurement of the pH values in the ER during ER stress and dexamethanose-induced stimulation.


Assuntos
Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Dexametasona/farmacologia , Transporte de Elétrons , Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio
8.
Chem Commun (Camb) ; 55(72): 10673-10676, 2019 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-31433407

RESUMO

We used single-molecule FRET experiments to monitor the conformational equilibrium of a DNA hairpin under pressure-stress in the presence of an aqueous two-phase system mimicking intracellular liquid-liquid phase separation (LLPS). Our data demonstrate the important role of LLPS with its immanent strong excluded volume effect in rescuing biological function under harsh environmental conditions.


Assuntos
DNA/química , Transferência Ressonante de Energia de Fluorescência , Conformação de Ácido Nucleico
9.
Chem Commun (Camb) ; 55(67): 9967-9970, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31367705

RESUMO

Herein, a seesaw ratiometric (SR) probe is designed which integrates fluorescence and surface enhanced Raman scattering (SERS) technology. Fluorescence imaging enables tracking of the spatiotemporal dynamic behaviour of telomerase. Meanwhile, SERS reverse ratiometric measurement can enable sensitive detection of telomerase activity in single living cells.


Assuntos
Corantes/química , Imagem Óptica/métodos , Análise de Célula Única/métodos , Análise Espectral Raman/métodos , Telomerase/metabolismo , DNA/química , Transferência Ressonante de Energia de Fluorescência/métodos , Ouro/química , Humanos , Células MCF-7 , Nanopartículas Metálicas/química
10.
Chem Commun (Camb) ; 55(61): 8963-8966, 2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31290488

RESUMO

We develop a simple method for sensitive detection of alkaline phosphatase (ALP) based on the ligase amplification reaction-catalyzed assembly of a single quantum dot (QD)-based nanosensor. This nanosensor requires only a single ligase enzyme to achieve ultrahigh sensitivity with a detection limit of 5.63 × 10-7 U mL-1, and can be applied for kinetic analysis, inhibitor screening, and ALP measurement in cell extracts.


Assuntos
Fosfatase Alcalina/análise , Técnicas Biossensoriais/métodos , DNA Ligases/química , Ensaios Enzimáticos/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Pontos Quânticos/química , Biotina/química , Carbocianinas/química , DNA/química , DNA/genética , Sondas de DNA/química , Sondas de DNA/genética , Fluorescência , Células HEK293 , Humanos , Limite de Detecção , Células MCF-7 , Hibridização de Ácido Nucleico , Imagem Individual de Molécula , Estreptavidina/química
11.
Phys Rev Lett ; 122(21): 218101, 2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31283336

RESUMO

Base-pair mismatch can relieve mechanical stress in highly strained DNA molecules, but how it affects their kinetic stability is not known. Using single-molecule fluorescence resonance energy transfer, we measured the lifetimes of tightly bent DNA loops with and without base-pair mismatch. Surprisingly, for loops captured by stackable sticky ends which leave single-stranded DNA breaks (or nicks) upon annealing, the mismatch decreased the loop lifetime despite reducing the overall bending stress, and the decrease was largest when the mismatch was placed at the DNA midpoint. These findings suggest that base-pair mismatch increases bending stress at the opposite side of the loop through an allosteric mechanism known as cooperative kinking. Based on this mechanism, we present a three-state model that explains the apparent dichotomy between thermodynamic and kinetic stability.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Transferência Ressonante de Energia de Fluorescência , Cinética , Modelos Químicos , Conformação de Ácido Nucleico , Termodinâmica
12.
Analyst ; 144(13): 3999-4005, 2019 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-31172144

RESUMO

Micrococcal nuclease (MNase) is an extracellular endonuclease of Staphylococcus aureus (S. aureus). It digests single stranded nucleic acid. The presence of MNase is the gold standard to identify S. aureus and its content. The present study reports the ultrahigh sensitive and selective fluorescence platform for MNase detection, designed and developed based on the surface energy transfer mechanism. A "proof of concept" is being developed based on monoclonal antibody-conjugated quantum dots (mAb-QDs), wherein mAb-QDs act as donors and graphene oxide (GO) acts as an acceptor. mAb-QDs in close proximity to GO undergo adsorption due to weak affinity between them and this results in fluorescence quenching by the transfer of surface energy from mAb-QDs to GO. During sensing, a much stronger affinity of mAb-QDs towards MNase inhibits the energy transfer to GO and this allows the regaining of fluorescence. Immobilized mAb-QDs on nitrocellulose membrane strips were fabricated and tested for "ON-OFF-ON" sensing of MNase. The limit of detection for fluorescence based assay and strips is found to be 0.3 ng mL-1 and 0.5 ng mL-1, respectively. The developed strips were applied on real samples for the detection of S. aureus.


Assuntos
Corantes Fluorescentes/química , Grafite/química , Nuclease do Micrococo/análise , Pontos Quânticos/química , Staphylococcus aureus/química , Anticorpos Monoclonais/química , Técnicas Biossensoriais/métodos , Colódio/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção , Membranas Artificiais , Nanocompostos , Estudo de Prova de Conceito , Sensibilidade e Especificidade , Staphylococcus aureus/enzimologia
13.
Adv Mater ; 31(33): e1901885, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31222950

RESUMO

Extracellular ATP is an emerging target for cancer treatment because it is a key messenger for shaping the tumor microenvironment (TME) and regulating tumor progression. However, it remains a great challenge to design biochemical probes for targeted imaging of extracellular ATP in the TME. A TME-driven DNA nanomachine (Apt-LIP) that permits spatially controlled imaging of ATP in the extracellular milieu of tumors with ultrahigh signal-to-background ratio is reported. It operates in response to the mild acidity in the TME with the pH (low) insertion peptide (pHLIP) module, thus allowing the specific anchoring of the structure-switching signaling aptamer unit to the membrane of tumor cells for "off-on" fluorescence imaging of the extracellular ATP. Apt-LIP allows for acidity driven visualization of different extracellular concentrations of exogenous ATP, as well as the monitoring of endogenous ATP release from cells. Furthermore, it is demonstrated that Apt-LIP represents a promising platform for the specific imaging of the extracellular ATP in both primary and metastatic tumors. Ultimately, since diverse aptamers are obtained through in vitro selection, this design strategy can be further applied for precise detection of various extracellular targets in the TME.


Assuntos
Trifosfato de Adenosina/metabolismo , Aptâmeros de Nucleotídeos/química , DNA/fisiologia , Nanoestruturas/química , Imagem Óptica/métodos , Microambiente Tumoral/fisiologia , Animais , Bacteriorodopsinas/química , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Humanos , Concentração de Íons de Hidrogênio , Camundongos , Peptídeos/química , Peptídeos/metabolismo
14.
Anal Bioanal Chem ; 411(21): 5481-5488, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31236648

RESUMO

The mechanism behind the variation in the photoluminescence (PL) of a MoS2 nanohybrid material with pH was investigated. Highly fluorescent MoS2 quantum dots dispersed across MoS2 nanosheets (MoS2 QDNS) were synthesized by a hydrothermal route in the presence of NaOH. Upon reducing the pH from 13 to 6.5, the PL intensity was markedly quenched. The removal of dangling sulfur atoms by adding mineral acids could be a plausible mechanism for this PL quenching, together with the inner filter effect and Förster resonance energy transfer due to the resulting species. A label-free turn-on fluorescence sensor for H2O2 was developed using this hybrid material. The PL of the acidified MoS2 QDNS at pH 6.5 increased (i.e., recovered) linearly with the concentration of H2O2. The dynamic range of the sensor was found to be 2-94 µM with a limit of detection (LOD) of 2 µM. This sensing strategy was also extended for the detection of glucose by appending glucose oxidase (GOx) as a catalyst. In the presence of GOx, glucose oxidizes to gluconic acid and H2O2, so the original level of glucose can be estimated by determining the H2O2 present. The absence of a complicated enzyme immobilization step is the prime advantage of the present glucose sensor. The current work exemplifies the utility of MoS2-based nanoparticle systems in the biological sensor domain. Graphical abstract.


Assuntos
Dissulfetos/química , Peróxido de Hidrogênio/análise , Concentração de Íons de Hidrogênio , Molibdênio/química , Transferência Ressonante de Energia de Fluorescência , Glucose Oxidase/análise , Limite de Detecção , Medições Luminescentes
15.
Int J Nanomedicine ; 14: 3571-3582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31213799

RESUMO

Background: In vivo fluorescence imaging in the second near-infrared (NIR-II, 1000-1700 nm) window using organic fluorophores has great advantages, but generally suffers from a relatively low fluorescence quantum yield (mostly less than 2%). In this study, organic nanoparticles (L1013 NPs) with a high fluorescence quantum yield (9.9%) were systhesized for in vivo imaging. Methods: A molecule (BTPPA) with donor-acceptor-donor structure and aggregation-induced emission enabling moieties was prepared. BTPPA molecules were then encapsulated into nanoparticles (L1013 NPs) using a nanoprecipitation method. The L1013 NPs were intravenously injected into the mice (including normal, stroke and tumor models) for vascular and tumor imaging. Results: L1013 NPs excited at 808 nm exhibit NIR-II emission with a peak at 1013 nm and an emission tail extending to 1400 nm. They have a quantum yield of 9.9% and also show excellent photo/colloidal stabilities and negligible in vitro and in vivo toxicity. We use L1013 NPs for noninvasive real-time visualization of mouse hindlimb and cerebral vessels (including stroke pathology) under a very low power density (4.6-40 mW cm‒2) and short exposure time (40-100 ms). Moreover, L1013 NPs are able to localize tumor pathology, with a tumor-to-normal tissue ratio of 11.7±1.3, which is unusually high for NIR-II fluorescent imaging through passive targeting strategy. Conclusion: L1013 NPs demonstrate the potential for a range of clinical applications, especially for tumor surgery.


Assuntos
Corantes Fluorescentes/química , Nanopartículas/química , Imagem Óptica/métodos , Espectroscopia de Luz Próxima ao Infravermelho , Animais , Vasos Sanguíneos/diagnóstico por imagem , Encéfalo/irrigação sanguínea , Encéfalo/diagnóstico por imagem , Modelos Animais de Doenças , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Camundongos Endogâmicos C57BL , Nanopartículas/toxicidade , Nanopartículas/ultraestrutura , Acidente Vascular Cerebral/diagnóstico por imagem , Distribuição Tecidual
16.
Chem Commun (Camb) ; 55(56): 8098-8101, 2019 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-31232416

RESUMO

We report the first macrocycle-based ratiometric molecular thermometer exploiting the conformational thermosensitivity of a calixarene functionalized with two different fluorophores. Thanks to the dependence on temperature of the efficiency of excitation energy transfer between the organic fluorophores, the thermometer works over a 60 °C-wide temperature range with a sensitivity of 4% °C-1.


Assuntos
Calixarenos/química , Corantes Fluorescentes/química , Temperatura Alta , Transferência Ressonante de Energia de Fluorescência , Compostos Macrocíclicos/química , Estrutura Molecular , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização por Electrospray
17.
Anal Chim Acta ; 1072: 75-80, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31146867

RESUMO

A highly specific and sensitive isothermal method for mercury detection using DNA-conjugated upconversion nanoparticles is reported. A single-stranded DNA containing thymine bases, used as the Hg2+-capturing element through the formation of thymine-Hg2+-thymine complex, is covalently attached to the NaYF4: Yb3+, Tm3+ nanoparticles. Luminescence resonance energy transfer takes place between the NaYF4: Yb3+, Tm3+ nanoparticles as donor and DNA-intercalating SYBR Green I as the acceptor upon excitation of 980 nm. The sensitivity and selectivity toward Hg2+ are enhanced using the nicking enzyme, Nt. Alwl, which leads to signal amplification. By monitoring the ratio of acceptor emission to a reference peak, the presence of Hg2+ ions are quantitatively determined with a lower detection limit of 0.14 nM, which is much lower than the US Environmental Protection Agency (EPA) limit of Hg2+ in drinking water.


Assuntos
DNA/química , Mercúrio/análise , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico , Água Potável/química , Transferência Ressonante de Energia de Fluorescência
18.
Phys Chem Chem Phys ; 21(24): 12843-12849, 2019 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-31179453

RESUMO

Methods based on fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) are widely used in the biological sciences, employing mostly dye-based FRET and PET pairs. While very useful and important, dye-based reporters are not always applicable without concern, for example, in cases where the fluorophore size needs to be minimized. Therefore, development and characterization of smaller, ideally amino acid-based PET and FRET pairs will expand the biological spectroscopy toolbox to enable new applications. Herein, we show that, depending on the excitation wavelength, tryptophan and 4-cyanotrptophan can interact with each other via the mechanism of either energy or electron transfer, hence constituting a dual FRET and PET pair. The biological utility of this amino acid pair is further demonstrated by applying it to study the end-to-end collision rate of a short peptide, the mode of interaction between a ligand and BSA, and the activity of a protease.


Assuntos
Triptofano/análogos & derivados , Triptofano/química , Transporte de Elétrons , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ligantes , Oligopeptídeos/química , Ligação Proteica , Soroalbumina Bovina/química , Espectrometria de Fluorescência , Tripsina/química
19.
Pestic Biochem Physiol ; 157: 108-121, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31153458

RESUMO

Karanjin, a furanoflavonol from Pongamia pinnata L is used in agricultural practices for its pesticidal, insecticidal and acaricidal activities. It is commercially available as a bio-pesticide targeting a wide variety of pests. The present study was intended to evaluate the biochemical interactions of karanjin with bovine serum albumin (BSA) and study its toxicological effects on mammalian and bacterial cell lines. Karanjin bound to BSA at a single site with a dissociation constant of 19.7 µM. Evaluation of BSA-karanjin interactions at three different temperatures indicated the involvement of static mode of quenching. Binding experiments in the presence of warfarin and computational docking analysis indicated that karanjin bound closer to the warfarin binding site located in the Subdomain IIA of BSA. Using Förster resonance energy transfer analysis the distance between TRP 213 of BSA and karanjin was found to be 20 Å. Collective results from synchronous fluorescence spectra analysis, differential scanning calorimetry, and circular dichroism analysis indicated that binding of karanjin induced conformational changes in the secondary structure of BSA. Karanjin exhibited low toxicity against human cervical cancer cells and normal mouse fibroblast L929 cells and modestly inhibited the growth of B. subtilis and E. coli cells. The data presented in this study provides insights for understanding the binding interactions of karanjin with BSA and its possible toxicological effects on mammalian cell lines and bacteria.


Assuntos
Benzopiranos/metabolismo , Benzopiranos/toxicidade , Óleos Vegetais/química , Óleos Vegetais/toxicidade , Pongamia/química , Sementes/química , Animais , Sítios de Ligação , Agentes de Controle Biológico/química , Agentes de Controle Biológico/toxicidade , Bovinos , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Transferência Ressonante de Energia de Fluorescência , Humanos , Camundongos , Ligação Proteica , Soroalbumina Bovina/metabolismo
20.
Chem Commun (Camb) ; 55(49): 7085-7088, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31150032

RESUMO

A luminescent biosensor has been developed for matrix metalloproteinase 9 (MMP-9) assays based on the selective interaction between an Ir(iii) solvent complex and a histidine-rich peptide, which avoids the complicated double labeling of substrate polypeptides commonly-used in FRET MMP detections and provides a promising strategy for MMP detection in clinical applications.


Assuntos
Técnicas Biossensoriais , Complexos de Coordenação/química , Histidina/química , Irídio/química , Metaloproteinase 9 da Matriz/análise , Peptídeos/química , Complexos de Coordenação/metabolismo , Transferência Ressonante de Energia de Fluorescência , Histidina/metabolismo , Humanos , Metaloproteinase 9 da Matriz/metabolismo , Peptídeos/metabolismo , Solventes/química
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