Seta-633 - A NIR Fluorescence Lifetime Label for Low-Molecular-Weight Analytes (original) (raw)
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Fluorescent lifetime of near infrared dyes for structural analysis of serum albumin
Progress in Biomedical Optics and Imaging - Proceedings of SPIE, 2008
Structural alteration of serum albumin, the major extracellular multifunctional protein in mammals, has been linked to a number of diseases. Herein we present a method based on fluorescence lifetime analysis of near-infrared (NIR) probes bound to albumin to interrogate its structural state without prior isolation of the protein. Molecular modeling study revealed that albumin binds polymethine dyes via two binding sites with different sizes and polarities. As a result, a NIR molecular probe typically exhibits two distinct lifetimes with corresponding fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture. To evaluate the effect of size and polarity of binding sites on fluorescence lifetime we studied NIR probes in solutions with different viscosity and polarity. We established that viscosity has negligible effect on polymethine dyes lifetime while the change in polarity has a profound impact. We also established a correlation between fluorescence lifetime and solvent polarity function for a number of NIR dyes for quantitative description of binding sites polarity. After screening a library of dyes, we identified a probe with optimal fluorescence lifetime properties to assess structure-related differences of albumins. The results show that changes in the lifetime of NIR dyes reflect the perturbation of albumin's tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes of albumins in whole blood.
Photochemistry and Photobiology, 2007
A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high-throughput structure-related analysis of albumin by using the fluorescence lifetime properties of near-infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structurerelated differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes in albumin in whole blood without prior isolation of the pure protein.
Fluorescent lifetime of near infrared dyes for structural analysis of serum albumin
Molecular Probes for Biomedical Applications II, 2008
Structural alteration of serum albumin, the major extracellular multifunctional protein in mammals, has been linked to a number of diseases. Herein we present a method based on fluorescence lifetime analysis of near-infrared (NIR) probes bound to albumin to interrogate its structural state without prior isolation of the protein. Molecular modeling study revealed that albumin binds polymethine dyes via two binding sites with different sizes and polarities. As a result, a NIR molecular probe typically exhibits two distinct lifetimes with corresponding fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture. To evaluate the effect of size and polarity of binding sites on fluorescence lifetime we studied NIR probes in solutions with different viscosity and polarity. We established that viscosity has negligible effect on polymethine dyes lifetime while the change in polarity has a profound impact. We also established a correlation between fluorescence lifetime and solvent polarity function for a number of NIR dyes for quantitative description of binding sites polarity. After screening a library of dyes, we identified a probe with optimal fluorescence lifetime properties to assess structure-related differences of albumins. The results show that changes in the lifetime of NIR dyes reflect the perturbation of albumin's tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes of albumins in whole blood.
Journal of Biomedical Optics, 2008
Fluorescence lifetime (FLT) information is complementary to intensity measurement and can be used to improve signal-to-background contrast and provide environment sensing capability. In this study, we evaluated the FLTs of eight near-infrared fluorescent molecular probes in vitro in various solvent mediums and in vivo to establish the correlation between the in vitro and in vivo results. Compared with other mediums, two exponential fitting of the fluorescence decays of dyes dissolved in aqueous albumin solutions accurately predicted the range of FLTs observed in vivo. We further demonstrated that the diffusion of NIR reporter from a dye-loaded gel can be detected by FLT change in mice as a model of controlled drug release. The mean FLT of the NIR probe increased as the dye diffused from the highly polar gel interior to the more lipophilic tissue environment. The two-point analysis demonstrates an efficient in vitro method for screening new NIR fluorescent reporters for use as FLT probes in vivo, thereby minimizing the use of animals for FLT screening studies.
New Fluorescence Probes for Biomolecules
Molecules, 2015
Steady state fluorescence measurements have been used for the investigation of interaction between the bovine serum albumin (BSA) and fluorescence probes: 3-hydroxy-2,4bis[(3-methyl-1,3-benzoxazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ6), 3-hydroxy-2,4-bis[(3-methyl-1,3-benzothiazol-2(3H)-ylidene)methyl]cyclobut-2-en-1-one (SQ7) and 3-hydroxy-2,4-bis[(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)methyl]cyclobut-2-en-1-one (SQ8). The binding constant between bovine serum albumin and squarine dyes has been determined by using both the Benesi-Hildebrand and Stern-Volmer equations. The negative value of free energy change indicates the existence of a spontaneous complexation process of BSA with squarine dyes.
Fluorescent Probes and Labels for Biomedical Applications
Annals of the New York Academy of Sciences, 2008
Fluorescence probes and labels have become indispensable tools for clinical diagnostics, highthroughput screening, and other biomedical applications. We have developed several classes of new squaraine-based red and near-infrared (NIR) probes and labels (SETA and Square series), naphthalimide-based fluorescence lifetime dyes (SeTau series), and cyanine-and squaraine-based quenchers (SQ series). This report discusses the spectral and photophysical properties of these new markers. In particular, the red and NIR dyes of the SETA and Square series are extremely bright, with photostabilities that are unmatched by any other dyes in the same spectral region.
Fluorometric assay for quantitation of biotin covalently attached to proteins and nucleic acids.
As a component of the (strept)avidin affinity system, biotin is often covalently linked to proteins or nucleic acids. We describe here a microplate-based high-throughput fluorometric assay for biotin linked to either proteins or nucleic acids based on fluorescence resonance energy transfer (FRET). This assay utilizes a complex of Alexa Fluoro 488 dye-labeled avidin with a quencher dye, 2-(4'-hydroxyazobenzene) benzoic acid (HABA), occupying the biotin binding sites of the avidin. In the absence of biotin, HABA quenches the fluorescence emission of the Alexa Fluor 488 dyes via FRET HABA is displaced when biotin binds to the Alexa Fluor 488 dye-labeled avidin, resulting in decreased FRET efficiency. This mechanism results in an increase in fluorescence intensity directly related to the amount of biotin present in the sample. The assay is able to detect as little as 4 pmol biotin in a 0.1 mL volume within 15 min of adding sample to the reagent, with a Z-factor > 0.9.
Journal of chromatography. B, Biomedical sciences and applications, 2001
This paper demonstrates the use of a near-infrared (NIR) dye as a non-covalent label for human serum albumin (HSA). The dye is a water soluble, heptamethine cyanine dye. The utility of the dye as a tracer illustrating the binding of various drugs to HSA is demonstrated via affinity capillary electrophoresis with near-infrared laser-induced fluorescence detection (ACE-NIR-LIF). Additionally, the factors affecting the separation of relevant species were investigated. The change in quantum yield of the dye upon complexation with HSA was calculated. Spectrophotometric measurements were conducted to study the stoichiometry of the dye albumin complex.
2017
Monoclonal antibodies labeled with near-infra-red (NIR) fluorophores have potential use in disease detection, intraoperative imaging, and pharmacokinetic characterization of therapeutic antibodies in both the preclinical and clinical setting. Recent work has shown conjugation of NIR fluorophores to antibodies can potentially alter antibody disposition at a sufficiently high degree of labeling (DoL); however, other reports show minimal impact after labeling with NIR fluorophores. In this work, we label two clinically approved antibodies, Herceptin (trastuzumab) and Avastin (bevacizumab), with NIR dyes IRDye 800CW (800CW) or Alexa Fluor 680 (AF680), at 1.2 and 0.3 dyes/antibody and examine the impact of fluorophore conjugation on antibody plasma clearance and tissue distribution. At 0.3 DoL, AF680 conjugates exhibited similar clearance to unlabeled antibody over 17 days while 800CW conjugates diverged after 4 days, suggesting AF680 is a more suitable choice for long-term pharmacokinetic studies. At the 1.2 DoL, 800CW conjugates cleared faster than unlabeled antibodies after several hours, in agreement with other published reports. The tissue biodistribution for bevacizumab−800CW and −AF680 conjugates agreed well with literature reported biodistributions using radiolabels. However, the greater tissue autofluorescence at 680 nm resulted in limited detection above background at low (∼2 mg/kg) doses and 0.3 DoL for AF680, indicating that 800CW is more appropriate for short-term biodistribution measurements and intraoperative imaging. Overall, our work shows a DoL of 0.3 or less for non-site-specifically labeled antibodies (with a Poisson distribution) is ideal for limiting the impact of NIR fluorophores on antibody pharmacokinetics.
Chemical communications (Cambridge, England), 2018
Developing sensitive and selective near-infrared fluorescent bio-probes for serum albumin detection is an ambitious and highly rewarding task. Herein, we report a styryl based fluorophore for serum albumin detection, which displays an exceptional turn-on emission enhancement of ∼500 fold, the highest reported so far in the near-infrared region, and more importantly enables quantification of albumin in the complex serum matrix.