Synthesis, spectral properties, and detection limits of reactive squaraine dyes, a new class of diode laser compatible fluorescent protein labels
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Journal Article↗
Biomedical subjects
H Szmacinski
Publications and source records attributed to H Szmacinski.
Lifetime-based pH sensors: indicators for acidic environments.
We characterized the pH-dependent intensity decays of three fluorophores, Oregon green 514 carboxylic acid, Cl-NERF, and DM-NERF, using frequency-domain fluorometry, with the objective of identifying lifetime-based sensors for low pH values. These three probes were originally designed as dual excitation wavelength-ratiometric probes, with high photostability and high quantum yields in aqueous solutions. We found that their fluorescence intensity decays were strongly dependent on pH. Moreover, global intensity decays analysis reveals that these probes have double exponential intensity decays at intermediate pH values and that the decay time amplitudes are greatly dependent on pH. The longer lifetime components originated from the unprotonated forms and the shorter components from the protonated forms. Both forms can emit fluorescence at intermediate pH values. The apparent pKa values were also determined from the titration curves of phase angles and modulations versus pH for the purpose of pH sensing. The apparent pKa values range from pH 3 to 5, a range where lifetime-based sensors are not presently reported. Since these probes show low pKa values and display substantial phase and modulation changes with pH, they are suitable as lifetime-based pH sensors to monitor the pH changes in acidic environments. One potential application of these probes is to trace the pH in different cellular compartments.
Fluoresceins↗
Long-lifetime Ru(II) complexes for the measurement of high molecular weight protein hydrodynamics.
We describe the synthesis and characterization of two asymmetrical ruthenium(II) complexes, [Ru(dpp)2(dcbpy)]2+ and [Ru(dpp)2(mcbpy)]2+, as well as the water soluble sulfonated derivatives [Ru(dpp(SO3Na)2)2(dcbpy)]2+ and [Ru(dpp(SO3Na)2)2(mcbpy)]2+ (dpp is 4,7-diphenyl-1,10-phenanthroline, dcbpy is 4,4'-dicarboxylic acid-2,2'-bipyridine, mcbpy is 4-methyl,4'-carboxylic acid-2,2'-bipyridine, and dpp(SO3Na)2 is the disulfonated derivative of dpp) as probes for the measurement of the rotational motions of proteins. The spectral (absorption, emission, and anisotropy) and photophysical (time-resolved intensity and anisotropy decays) properties of these metal-ligand complexes were determined in solution, in both the presence and absence of human serum albumin (HSA). These complexes display lifetimes ranging from 345 ns to 3.8 microseconds in deoxygenated aqueous solutions under a variety of conditions. The carboxylic acid groups on these complexes were activated to form N-hydroxysuccinimide (NHS) esters which were used to covalently lable HSA, and were characterized spectroscopically in the same manner as above. Time-resolved anisotropy measurements were performed to demonstrate the utility of these complexes in measuring long rotational correlation times of bioconjugates between HSA and antibody to HSA. The potential usefulness of these probes in fluorescence polarization immunoassays was demonstrated by an association assay of the Ru(II)-labeled HSA with polyclonal antibody.
Anisotropy↗
Spatially localized ballistic two-photon excitation in scattering media.
We describe spatially localized two-photon excitation in scattering media. Using femtosecond pulses at 770 nm from a Ti: Sapphire laser, we were able to excite fluorophores in capillary tubes under up to 1.5 mm of 0.5% intralipid. Displacement of the laser beam relative to the embedded samples indicates that highly localized excitation was possible with two-photon excitation, whereas one-photon excitation resulted in loss of spatial resolution due to excitation by the diffusely scattered photons. These results indicate that two-photon excitation in the scattering solution is due only to the ballistic photons, a result confirmed by frequency-domain time-resolved measurements. Selective excitation of adjacent embedded samples was found possible for two but not one-photon excitation.
Fat Emulsions, Intravenous↗
A long-lived, highly luminescent Re(I) metal-ligand complex as a biomolecular probe.
A highly luminescent rhenium (I) metal-ligand complex [Re(bcp)(CO)3(4-COOHPy)](ClO4), where bcp is 2,9-dimethyl-4,7-diphenyl-1,10-phenanthroline and 4-COOHPy is isonicotinic acid, has been synthesized and characterized. High quantum yields (> 0.5) and long excited-state lifetimes (0.3-10 micronseconds) in fluid solutions at room temperature were found for this complex, with remarkable emission sensitivity to microenvironment. This compound also displays highly polarized emission with a maximum anisotropy near 0.3 in the absence of rotational diffusion. This Re complex was conjugated to several biomolecules, including the proteins human serum albumin and bovine immunoglobulin G, as well as an amine-containing lipid. When bound to a protein or lipid, the decay time is near 3 microseconds and the quantum yield is approximately 0.12 in aqueous oxygenated solution at room temperature. This compound's unique spectral properties along with its conjugatability allowed us to utilize it as biomolecular probe in a variety of environments.
Anisotropy↗
Synthesis and spectral characterization of a thiol-reactive long-lifetime Ru(II) complex.
We report the synthesis and spectral properties of a long-lifetime luminescent Ru complex containing a sulfhydryl-reactive maleimide group, [Ru (2,2'-bipyridine)2(1, 10-phenanthroline-5-maleimide)](PF6)2. [Ru(bpy)2(phen-mi)]2+ was covalently linked to human serum albumin, immunoglobulin G, and beta-galactosidase. The lifetimes for probe bound to proteins were near 1.1 micros. In the absence of rotational motions, the probe displayed an anisotropy near 0.17 for excitation near 475 nm. Anisotropy decay data were used to determine rotational correlation times of the proteins, which showed local probe motions in addition to overall rotational diffusion. This long-lifetime sulfhydryl-reactive probe can be used to recover microsecond rotational motions and/or domain motions of proteins and/or macromolecular complexes.
Fluorescence Polarization Immunoassay↗
Sodium Green as a potential probe for intracellular sodium imaging based on fluorescence lifetime.
We characterized the use of the fluorescent probe Sodium Green for measurements of intracellular free sodium using frequency-domain, phase-modulation fluorometry. The intensity decays were found to be strongly Na+ dependent, with mean lifetime increasing from 1.13 ns in the absence of Na+ to 2.39 ns in the presence of 140 mM Na+. Detailed analysis of the intensity decays in the presence of Na+ and K+ in the concentration range from 0 to 500 mM is provided. Sodium sensing using data measured at a single modulation frequency is described. Phase and modulation data showed high sensitivity to Na+ and substantially lower sensitivity to K+. Additionally, exposure of Sodium Green to intense illumination indicated that Sodium Green is much more photostable than its precursor, fluorescein. These results indicate that lifetime-based measurements with Sodium Green can be used for imaging of intracellular free [Na+] in the range from about 0.5 to 50 mM with high accuracy.
Flow Cytometry↗
Lifetime-based sensing of glucose using energy transfer with a long lifetime donor.
We describe an optical assay for glucose based on the luminescence decay time of a long lifetime metal-ligand complex. Concanavalin A was covalently labeled with Ruthenium metal-ligand complex (RuCon A) which served as the donor. The acceptor was malachite green which was covalently linked to insulin. The malachite green insulin was also covalently labeled with maltose (MIMG) to provide binding affinity to RuCon A. Binding of RuCon A to MIMG resulted in a decreased intensity and decay time of RuCon A. Glucose was detected by competitive displacement of MIMG from RuCon A, resulting in increased intensity and decay time. This glucose assay has several favorable features. The long lifetime of RuCon A allows phase-modulation decay time measurements using an amplitude-modulated bluelight-emitting diode as the light source. Reversibility of the assay can be controlled by the extent of sugar labeling of the insulin. Finally, the glucose-sensitive range can be adjusted by selection of the sugar structure and extent of labeling of the insulin.
Blood Glucose↗
Synthesis and luminescence spectral characterization of long-lifetime lipid metal-ligand probes.
We synthesized phospholipid analogues of phosphatidyl ethanolamine which contains a ruthenium metal-ligand complex (MLC) covalently bound to the amino group. Two analogues were synthesized, containing either one (Ru-PE) or two (Ru-PE2) lipid molecules covalently linked to the MLC by the amino group of the lipid. These MLC-lipid probes display intensity decay times from 682 to 357 ns, depending on temperature. Importantly, the luminescence MLC groups display polarized emission, enabling their use for studies of membrane dynamics. The long intensity decay times allowed measurement of the overall rotation correlation time of lipid vesicles to several microseconds. The spectral properties of the model membranes containing Ru-PE or Ru-PE2 were independent of the probe-to-lipid molar ratio from 1:20 to 1:100, suggesting minimal tendency for probe-probe interactions. These MLC-lipid probes can be expected to have numerous applications in studies of membrane dynamics on the microsecond timescale.
2,2'-Dipyridyl↗
Long-lifetime metal-ligand complexes as probes in biophysics and clinical chemistry.
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Animals↗
Synthesis and evaluation of Ru-complexes as anisotropy probes for protein hydrodynamics and immunoassays of high-molecular-weight antigens.
We investigated three unsymmetrical Ru-complexes, namely [Ru(bpy)2 (phen-ITC)]2+, [Ru(bpy)2(dcbpy)] and [Ru(bpy)2(mcbpy)]+ for use as probes for rotational diffusion and in immunoassays of high-molecular-weight antigens. For this purpose we synthesized reactive forms of these metal-ligand complexes and conjugated them to human serum albumin (HSA). The maximal anisotropies (r0) for the HSA-bound forms in frozen solution are 0.23, 0.17 and 0.14 for the (dcbpy), (mcbpy) and (phen-ITC) derivatives, respectively. The activated Ru metal-ligand complexes have either one or two NHS-esters or an isothiocyanate group as the reactive moiety. The usefulness of these complexes in immunoassays was determined by titration of the labeled HSA with polyclonal anti-HSA. The highest steady state anisotropy (r) values (0.190) were observed for the [Ru(bpy)2(dcbpy)]-labeled HSA on titration with polyclonal antibody. However, a relative increase in the steady state anisotropy (r/r0) on titration with polyclonal antibody was found for the phen-ITC probe (96%), as compared to the dcbpy (83%) or mcbpy (79%) derivatives. These findings were confirmed by time-resolved frequency-domain measurements. In particular the higher mean correlation times calculated for the phen-ITC derivative suggests reduced local probe motion for this probe when bound to HSA as compared to the (mcbpy) and (dcbpy) conjugates.
Anisotropy↗
Fluorescence polarization immunoassay of a high-molecular-weight antigen using a long wavelength-absorbing and laser diode-excitable metal-ligand complex.
We describe a fluorescence polarization immunoassay (FPI) based on an osmium metal-ligand complex, Os(bpy)2(dcbpy). The Os complex was selected for its long wavelength absorption, which allows excitation with wavelengths up to 720 nm. At these wavelengths tissue absorbance and autofluorescence are minimal, and excitation can be accomplished with LEDs or laser diodes. The Os complex displays a lifetime of 19 ns and a high initial anisotropy at two excitation wavelengths, 505 and 690 nm. A reactive NHS-ester of Os- (bpy)2(dcbpy) was synthesized containing a NHS ester, and used to label the human serum albumin (HSA) or anti-HSA. The FPIs of HSA were performed with monoclonal and polyclonal antibodies using excitation at 505 or 685 nm. The results showed the potential of the Os-ligand complex to be used as a long excitation wavelength FPI probe for potential use in homogeneous immunoassays with simple excitation sources.
2,2'-Dipyridyl↗
Distance distributions from the tyrosyl to disulfide residues in the oxytocin and [Arg8]-vasopressin measured using frequency-domain fluorescence resonance energy transfer.
We have examined the fluorescence intensity decays of oxytocin and [Arg8]-vasopressin resulting from the single tyrosyl residue in each peptide, and the intensity decay of the Asu1,6-analogues in which the disulfide bridge is substituted by a CH2-CH2 bridge. Viscosity-dependent steady state and intensity decay measurements indicated that fluorescence resonance energy transfer (FRET) from tyrosyl phenol to the disulfide bridge is responsible for the decrease in fluorescence relative to the Asu-analogues. The frequency-domain phase and modulation data for the tyrosyl donor were interpreted in terms of fluorescence resonance energy transfer (FRET) to the weakly absorbing disulfide bridge and a distribution of donor-to-acceptor distances. Energy transfer efficiencies were determined from both time-resolved and steady-state measurements. Fitting the frequency-domain phase and modulation data to a Gaussian distance distribution indicated that the average inter-chromophoric distance (Rav) is similar in both compounds, Rav = 7.94 A for oxytocin and Rav = 8.00 A for vasopressin. However, the width of the distance distribution is narrower for vasopression (hw = 2.80 A) than for oxytocin (hw = 3.58 A), which is consistent with restriction of the tyrosine phenol motion due to its stacking wih the Phe3 side chain of vasopressin. Finally, the recovered distance distribution functions are compared with histograms describing the distance between the chromophores during the course of long, in vacuo, molecular dynamics runs using the computer program CHARMm and the QUANTA 3.0 parameters.
Amino Acid Sequence↗
Three-photon induced fluorescence of the calcium probe Indo-1.
We report the calcium-dependent emission spectral properties of the calcium probe Indo-1 for three-photon excitation. We found that Indo-1 could be readily excited with the femtosecond pulses from a mode-locked Ti:sapphire laser at 885 nm. This wavelength is too long for two-photon excitation, which is expected to occur for wavelengths no longer than twice the longest single-photon absorption wavelength of 400 nm. For excitation at 885 nm the emission intensity was found to depend on the cube of the laser power, as expected for simultaneous interaction with three photons. At wavelengths below 840 nm the emission intensity depends on the square of the laser power, indicating two-photon excitation at shorter wavelengths. The intensity decays of Indo-1 were found to be dependent on Ca2+ and essentially identical for one- and three-photon excitation. The emission anisotropy of Indo-1 was found to be considerably higher for three-photon excitation than for one-photon excitation, consistent with cos6 theta photoselection, as compared with cos2 theta photoselection for one-photon excitation. The high values of the anisotropy are in agreement with those expected for a three-photon process. Calcium-dependent emission spectra were observed for Indo-1 with three-photon excitation, demonstrating that three-photon excitation of Indo-1 can be used for calcium imaging by emission intensity ratio measurements. The calcium-dependent emission spectra indicate a higher three-photon cross-section for the calcium-free form of Indo-1 than for the calcium-bound form. The possible advantages of three-photon excitation include the availability of the appropriate wavelengths with solid-state lasers, enhanced spatial resolution due to a reduced size of the excited volume, absence of light quenching, and possibly high selectivity of the three-photon excitation process.
Biophysical Phenomena↗
Fluorescence energy transfer immunoassay based on a long-lifetime luminescent metal-ligand complex.
We describe an immunoassay based on fluorescence resonance energy transfer (FRET). The antigen was human serum albumin (HSA), which was labeled with a ruthenium-ligand complex, [Ru(bpy)2(phen-ITC)]2+. The antibody (IgG) to HSA was labeled with a nonfluorescent absorber, Reactive Blue 4. Association of the Ru-labeled HSA with the antibody was detected by three spectral parameters, a decreased quantum yield of Ru-HSA, a decrease in its fluorescence lifetime, and an increase in its fluorescence anisotropy. The steady-state anisotropy of Ru-HSA increased approximately eightfold upon binding to the antibody. These spectral effects were observed both in the direct association of the Ru-HSA with Reactive Blue 4-labeled antibody, and in a competitive assay format wherein unlabeled HSA competed with Ru-HSA for the binding sites on the antibody. Some nonspecific interactions of HSA may have occurred with Reactive Blue 4-labeled AHA, a difficulty which can be avoided with a different acceptor. The use of FRET provides a reliable means to alter the spectral properties upon antigen-antibody binding. The advantages of a ruthenium-ligand fluorophore include its long-wavelength absorption and emission, long fluorescence lifetime, and high photo-stability. Long wavelengths minimize problems of autofluorescence from biological samples, and long life-times allow off-gating of the prompt autofluorescence.
Antigen-Antibody Complex↗
Fluorescence polarization immunoassay of a high-molecular-weight antigen based on a long-lifetime Ru-ligand complex.
We describe a new class of fluorescence polarization immunoassays based on the luminescence from an asymmetrical Ru-ligand complex. We found that such a complex displays larger polarization values than those of comparable symmetrical complexes and appear to be highly photostable in aqueous solution. We synthesized a conjugatable Ru-ligand complex, which was used to label human serum albumin (HSA) as the antigen. The Ru-ligand complex displays a long decay time near 400 ns when covalently linked to proteins. We found that the steady-state polarization of labeled HSA was sensitive to binding of anti-HSA, resulting in a 200% increase in polarization. The labeled HSA was also used in a competitive format using unlabeled HSA as the antigen. The time-resolved anisotropy decays demonstrate increased correlation times for labeled HSA in the presence of anti-HSA, an effect which was partially reversed in the presence of unlabeled HSA. These results demonstrate the potential of the metal-ligand complexes to be used in the fluorescence polarization immunoassay of high-molecular-weight analytes. The use of such metal-ligand complexes enable fluorescence polarization immunoassays which bypass the usual limitation to low-molecular-weight antigens, which is a consequence of the 2-5 ns decay time of the previously used fluorophores.
2,2'-Dipyridyl↗
Possibility of simultaneously measuring low and high calcium concentrations using Fura-2 and lifetime-based sensing.
We characterized the fluorescence probe Fura-2 for calcium measurements using frequency-domain phase-modulation fluorometry. By the use of different excitation wavelengths from 345 to 380 nm, the apparent calcium dissociation constants can be altered from 41 nM to 1.92 microM Ca2+. This change in apparent Kd results from changes in the relative extent of excitation of the calcium-bound and calcium-free forms, and the excitation wavelength-dependent contribution of each form to the intensity decay. These results indicate that lifetime-based measurements with Fura-2 can be used for imaging of calcium over a wide range of concentrations. An additional favorable feature of Fura-2 is that the calcium-free form can be almost exclusively excited at wavelength of 390 nm or longer, and can thus be used as a reference providing the lifetime in the absence of calcium, without removing the calcium. Additionally, exposure of Fura-2 to intense illumination shifts but does not distort the frequency response. For cellular imaging, these favorable properties of Fura-2 may allow calibration of the calcium concentrations without the use of ionophores.
Calcium↗
Metal-ligand complexes as a new class of long-lived fluorophores for protein hydrodynamics.
We describe the use of asymmetric Ru-ligand complexes as a new class of luminescent probes that can be used to measure rotational motions of proteins. These complexes are known to display luminescent lifetimes ranging from 10 to 4000 ns. In this report, we show that the asymmetric complex Ru(bpy)2(dcbpy) (PF6)2 displays a high anisotropy value when excited in the long wavelength absorption band. For covalent linkage to proteins, we synthesized the N-hydroxy succinimide ester of this metal-ligand complex. To illustrate the usefulness of these probes, we describe the intensity and anisotropy decays of [Ru(bpy)2(dcbpy)] when covalently linked to human serum albumin, concanavalin A (ConA), human immunoglobulin G (IgG), and Ferritin, and measured in solutions of increased viscosity. These data demonstrate that the probes can be used to measure rotational motions on the 10 ns to 1.5 microseconds timescale, which so far has been inaccessible using luminescence methods. The present probe [Ru(bpy)2(dcbpy)] can be regarded as the first of a class of metal-ligand complexes, each with different chemical reactivity and spectral properties, for studies of macromolecular dynamics.
Biophysical Phenomena↗