RESUMO
Current practical methods for finding the equilibrium dissociation constant, Kd , of protein-small molecule complexes have inherent sources of inaccuracy. Introduced here is "accurate constant via transient incomplete separation" (ACTIS), which appears to be free of inherent sources of inaccuracy. Conceptually, a short plug of the pre-equilibrated protein-small molecule mixture is pressure-propagated in a capillary, causing fast transient incomplete separation of the complex from the unbound small molecule. A superposition of signals from these two components is measured near the capillary exit and used to calculate a fraction of unbound small molecule, which, in turn, is used to calculate Kd . Herein the validity of ACTIS is proven theoretically, its accuracy is verified by computer simulation, and its practical use is demonstrated. ACTIS has the potential to become a reference-standard method for determining Kd â values of protein-small molecule complexes.
Assuntos
Eletroforese Capilar/métodos , Proteínas/químicaRESUMO
The limit-of-detection (LOD) in analytical instruments with fluorescence detection can be improved by reducing noise of optical background. Efficiently reducing optical background noise in systems with spectrally nonuniform background requires complex optimization of an emission filter-the main element of spectral filtration. Here, we introduce a filter-optimization method, which utilizes an expression for the signal-to-noise ratio (SNR) as a function of (i) all noise components (dark, shot, and flicker), (ii) emission spectrum of the analyte, (iii) emission spectrum of the optical background, and (iv) transmittance spectrum of the emission filter. In essence, the noise components and the emission spectra are determined experimentally and substituted into the expression. This leaves a single variable-the transmittance spectrum of the filter-which is optimized numerically by maximizing SNR. Maximizing SNR provides an accurate way of filter optimization, while a previously used approach based on maximizing a signal-to-background ratio (SBR) is the approximation that can lead to much poorer LOD specifically in detection of fluorescently labeled biomolecules. The proposed filter-optimization method will be an indispensable tool for developing new and improving existing fluorescence-detection systems aiming at ultimately low LOD.
RESUMO
Accurate quantitation of microRNA (miRNA) in tissue samples is required for validation and clinical use of miRNA-based disease biomarkers. Since sample processing, such as RNA extraction, introduces undesirable biases, it is advantageous to measure miRNA in a crude cell lysate. Here, we report on accurate miRNA quantitation in crude cell lysate by a CE-based hybridization assay termed direct quantitative analysis of multiple miRNAs (DQAMmiR). Accuracy and precision of miRNA quantitation were determined for miRNA samples in a crude cell lysate, RNA extract from the lysate, and a pure buffer. The results showed that the measurements were matrix-independent with inaccuracies of below 13% from true values and relative standard deviations of below 11% from the mean values in a miRNA concentration range of 2 orders of magnitude. We compared DQAMmiR-derived results with those obtained by a benchmark miRNA-quantitation method-quantitative reverse transcription-polymerase chain reaction (qRT-PCR). qRT-PCR-based measurements revealed multifold inaccuracies and relative standard deviations of up to 70% in crude cell lysate. Robustness of DQAMmiR to changes in sample matrix makes it a perfect candidate for validation and clinical use of miRNA-based disease biomarkers.
Assuntos
Eletroforese Capilar/métodos , MicroRNAs/análise , Reação em Cadeia da Polimerase em Tempo Real , Sondas de DNA/metabolismo , Humanos , Células MCF-7 , MicroRNAs/metabolismo , Hibridização de Ácido NucleicoRESUMO
Direct quantitative analysis of multiple miRNAs (DQAMmiR) utilizes CE with fluorescence detection for fast, accurate, and sensitive quantitation of multiple miRNAs. Here we report on achieving single-nucleotide specificity and, thus, overcoming a principle obstacle on the way of DQAMmiR becoming a practical miRNA analysis tool. In general, sequence specificity is reached by raising the temperature to the level at which the probe-miRNA hybrids with mismatches melt while the matches remain intact. This elevated temperature is used as the hybridization temperature. Practical implementation of this apparently trivial approach in DQAMmiR has two major challenges. First, melting temperatures of all mismatched hybrids should be similar to each other and should not reach the melting temperature of any of the matched hybrids. Second, the elevated hybridization temperature should not deteriorate CE separation of the hybrids from the excess probes and the hybrids from each other. The second problem is further complicated by the reliance of separation in DQAMmiR on single-strand DNA binding protein (SSB) whose native structure and binding properties may be drastically affected by the elevated temperature. These problems were solved by two approaches. First, locked nucleic acid (LNA) bases were incorporated into the probes to normalize the melting temperatures of all target miRNA hybrids allowing for a single hybridization temperature; binding of SSB was not affected by LNA bases. Second, a dual-temperature CE was developed in which separation started with a high capillary temperature required for proper hybridization and continued at a low capillary temperature required for quality electrophoretic separation of the hybrids from excess probes and the hybrids from each other. The developed approach was sufficiently robust to allow its integration with sample preconcentration by isotachophoresis to achieve a limit of detection below 10 pM.
Assuntos
MicroRNAs/análise , MicroRNAs/genética , Proteínas de Ligação a DNA/química , Eletroforese Capilar , Fluorescência , Oligonucleotídeos/química , Especificidade por Substrato , TemperaturaRESUMO
A nanosecond laser near-infrared spectrometer was used to study singlet oxygen ((1)O2) emission in a protein matrix. Myoglobin in which the intact heme is substituted by Zn-protoporphyrin IX (ZnPP) was employed. Every collision of ground state molecular oxygen with ZnPP in the excited triplet state results in (1)O2 generation within the protein matrix. The quantum yield of (1)O2 generation was found to be equal to 0.9 ± 0.1. On the average, six from every 10 (1)O2 molecules succeed in escaping from the protein matrix into the solvent. A kinetic model for (1)O2 generation within the protein matrix and for a subsequent (1)O2 deactivation was introduced and discussed. Rate constants for radiative and nonradiative (1)O2 deactivation within the protein were determined. The first-order radiative rate constant for (1)O2 deactivation within the protein was found to be 8.1 ± 1.3 times larger than the one in aqueous solutions, indicating the strong influence of the protein matrix on the radiative (1)O2 deactivation. Collisions of singlet oxygen with each protein amino acid and ZnPP were assumed to contribute independently to the observed radiative as well as nonradiative rate constants.
Assuntos
Luminescência , Mioglobina/química , Processos Fotoquímicos , Protoporfirinas/química , Oxigênio Singlete/química , Algoritmos , Animais , Cavalos , Cinética , Lasers , Modelos Moleculares , Oxigênio/química , Teoria Quântica , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Água/químicaRESUMO
The photophysics of free pyrenedicarboxamide (Py-DCA) in solution as well as of single-stranded and double-stranded oligonucleotides (ss and ds ONs) containing 1-7 pyrene building blocks per strand were studied by steady-state and time-resolved fluorescence spectroscopy. It was found that the fluorescence quantum yield Phi(F) of free Py-DCA chromophore in solution is rather high (Phi(F) = 0.44). However, after incorporation of the chromophore into a ss ON the monomeric chromophore fluorescence is quenched more than 40-fold due to electron-transfer reactions with ON bases. An increase of the number n of neighboring pyrenes in an ON results in Phi(F) growth up to 0.25 at n = 6. Starting from n = 2, all fluorescence belongs mainly to excimer formed by pyrene chromophores. Sections composed of multiple pyrenes may be considered as robust functional entities that may serve as independent modules in DNA-based, functional nano-architectures.
Assuntos
DNA/química , Nanotecnologia , Pirenos/química , Absorção , Amidas/química , Sequência de Bases , DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Capillary electrophoresis with Laser-Induced Fluorescence (CE-LIF) detection is being applied to new analytical problems which challenge both the power of CE separation and the sensitivity of LIF detection. On-capillary LIF detection is much more practical than post-capillary detection in a sheath-flow cell. Therefore, commercial CE instruments utilize solely on-capillary CE-LIF detection with a Limit of Detection (LOD) in the nM range, while there are multiple applications of CE-LIF that require pM or lower LODs. This tutorial analyzes all aspects of on-capillary LIF detection in CE in an attempt to identify means for improving LOD of CE-LIF with on-capillary detection. We consider principles of signal enhancement and noise reduction, as well as relevant areas of fluorophore photochemistry and fluorescent microscopy.
RESUMO
Photodynamic therapy that uses photosensitizers which only become toxic upon light-irradiation provides a strong alternative to conventional cancer treatment due to its ability to selectively target tumour material without affecting healthy tissue. Transition metal complexes are highly promising PDT agents due to intense visible light absorption, yet the majority are toxic even without light. This study introduces a small, photostable, charge-neutral platinum-based compound, Pt(II) 2,6-dipyrido-4-methyl-benzenechloride, complex 1, as a photosensitizer, which works under visible light. Activation of the new photosensitizer at low concentrations (0.1-1 µM) by comparatively low dose of 405 nm light (3.6 J cm(-2)) causes significant cell death of cervical, colorectal and bladder cancer cell lines, and, importantly, a cisplatin resistant cell line EJ-R. The photo-index of the complex is 8. We demonstrate that complex 1 induces irreversible DNA single strand breaks following irradiation, and that oxygen is essential for the photoinduced action. Neither light, nor compound alone led to cell death. The key advantages of the new drug include a remarkably fast accumulation time (diffusion-controlled, minutes), and photostability. This study demonstrates a highly promising new agent for photodynamic therapy, and attracts attention to photostable metal complexes as viable alternatives to conventional chemotherapeutics, such as cisplatin.
Assuntos
Antineoplásicos/farmacologia , Fotoquimioterapia/métodos , Fármacos Fotossensibilizantes/farmacologia , Platina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Humanos , LigantesRESUMO
As various fullerene derivatives have been developed, it is necessary to explore their photophysical properties for potential use in photoelectronics and medicine. Here, we address the photophysical properties of newly synthesized water-soluble fullerene-based nanoparticles and polyhydroxylated fullerene as a representative water-soluble fullerene derivative. They show broad emission band arising from a wide-range of excitation energies. It is attributed to the optical transitions from disorder-induced states, which decay in the nanosecond time range. We determine the kinetic properties of the singlet oxygen ((1)O2) luminescence generated by the fullerene nanoparticles and polyhydroxylated fullerene to consider the potential as photodynamic agents. Triplet state decay of the nanoparticles was longer than (1)O2 lifetime in water. Singlet oxygen quantum yield of a series of the fullerene nanoparticles is comparably higher ranging from 0.15 to 0.2 than that of polyhydroxylated fullerene, which is about 0.06.
Assuntos
Fulerenos/química , Nanopartículas/química , Fármacos Fotossensibilizantes/química , Oxigênio Singlete/química , Hidroxilação , Cinética , Luz , Luminescência , Medições Luminescentes , Oxirredução , Processos Fotoquímicos , Teoria Quântica , Oxigênio Singlete/análise , Solubilidade , Soluções/efeitos da radiação , Termodinâmica , Fatores de Tempo , Água/químicaRESUMO
A linear asymmetric Pt(ii) trans-acetylide donor-bridge-acceptor triad designed for efficient charge separation, NAP[triple bond, length as m-dash]Pt(PBu3)2[triple bond, length as m-dash]Ph-CH2-PTZ (), containing strong electron acceptor and donor groups, 4-ethynyl-N-octyl-1,8-naphthalimide (NAP) and phenothiazine (PTZ) respectively, has been synthesised and its photoinduced charge transfer processes characterised in detail. Excitation with 400 nm, â¼50 fs laser pulse initially populates a charge transfer manifold stemming from electron transfer from the Pt-acetylide centre to the NAP acceptor and triggers a cascade of charge and energy transfer events. A combination of ultrafast time-resolved infrared (TRIR) and transient absorption (TA) spectroscopies, supported by UV-Vis/IR spectroelectrochemistry, emission spectroscopy and DFT calculations reveals a self-consistent photophysical picture of the excited state evolution from femto- to milliseconds. The characteristic features of the NAP-anion and PTZ-cation are clearly observed in both the TRIR and TA spectra, confirming the occurrence of electron transfer and allowing the rate constants of individual ET-steps to be obtained. Intriguingly, has three separate ultrafast electron transfer pathways from a non-thermalised charge transfer manifold directly observed by TRIR on timescales ranging from 0.2 to 14 ps: charge recombination to form either the intraligand triplet (3)NAP with 57% yield, or the ground state, and forward electron transfer to form the full charge-separated state (3)CSS ((3)[PTZ(+)-NAP(-)]) with 10% yield as determined by target analysis. The (3)CSS decays by charge-recombination to the ground state with â¼1 ns lifetime. The lowest excited state is (3)NAP, which possesses a long lifetime of 190 µs and efficiently sensitises singlet oxygen. Overall, molecular donor-bridge-acceptor triad demonstrates excited state branching over 3 different pathways, including formation of a long-distant (18 Å) full charge-separated excited state from a directly observed vibrationally hot precursor state.
RESUMO
Time-resolved luminescence measurements in the near-infrared region indicate that photodissociation of molecular oxygen from myoglobin and hemoglobin does not produce detectable quantities of singlet oxygen. A simple and highly sensitive method of luminescence quantification is developed and used to determine the upper limit for the quantum yield of singlet oxygen production. The proposed method was preliminarily evaluated using model data sets and confirmed with experimental data for aqueous solutions of 5,10,15,20-tetrakis(4-N-methylpyridyl) porphyrin. A general procedure for error estimation is suggested. The method is shown to provide a determination of the integral luminescence intensity in a wide range of values even for kinetics with extremely low signal-to-noise ratio. The present experimental data do not deny the possibility of singlet oxygen generation during the photodissociation of molecular oxygen from myoglobin and hemoglobin. However, the photodissociation is not efficient to yield singlet oxygen escaped from the proteins into the surrounding medium. The upper limits for the quantum yields of singlet oxygen production in the surrounding medium after the photodissociation for oxyhemoglobin and oxymyoglobin do not exceed 3.4×10(-3) and 2.3×10(-3), respectively. On the average, no more than one molecule of singlet oxygen from every hundred photodissociated oxygen molecules can succeed in escaping from the protein matrix.