The luminescence characteristics of six osmium carbonyl complexes with phenanthroline (phen) or bipyridine (bpy) and pyridine (py), 4-phenylpyridine (4-phpy), or triphenylphosphine (PPh3) complexes in the presence of polyanion heparin were studied in both ethanol and aqueous solutions. The influence of heparin on the luminescence of the complexes is heavily dependent on the type of ligands in the complexes and the solvent used. In the ethanol solutions, the heparin solution enhanced the luminescence of the five osmium complexes, with the strongest enhancement to the 4-phenylpyridine complexes; linear curves were obtained in the luminescence enhancement ratio (
Heparina, a highly sulfated glycosaminoglycan, has the highest negative charge density of any known natural biological molecule [
Luminescence spectroscopy is an analytical method that studies the luminescence emission from a chromophore after their electrons are excited from the ground state to a higher energy level. One of the most attractive features of luminescence spectroscopy as an analytical method is its inherent high sensitivity, wide linear response range, and fast detection speed [
Recently, a series of Os(II) carbonyl (CO) complexes with two bipyridine or phenanthroline ligands and one pyridyl or triphenylphosphine ligand as the five other ligands have been synthesized in our lab. The osmium centers in these complexes are all in the +2 oxidation state with an overall +2 charge of the complex ion, and exhibit high emission intensity in the visible region [
The six new Os(II) complexes are [Os(phen)2CO(PPh3)](PF6)2, [Os(phen)2CO(py)](PF6)2, [Os(phen)2CO(4-phpy)](PF6)2, [Os(bpy)2CO(PPh3)](PF6)2, [Os(bpy)2CO(py)](PF6)2, and [Os(bpy)2CO(4-phpy)](PF6)2. The structure of each complex is shown in Figure
Structures of the six osmium complexes.
Heparin sodium sulfate (>180 USP units/mg), ethanol (99.5+%), reagent or analytical grade NaClO4, MgSO4, CH3COONa, NaCl, KBr, NaNO3, NaOH, NaNO2, KSCN, and KI were all obtained from Sigma-Aldrich. Injectable heparin solutions, 100 units/mL and 40 units/mL with 5% dextrose, were purchased from B. Braun Medical Inc. (Irving, CA, USA).
Luminescence measurements were made on a Perkin Elmer luminescence spectrometer LS 50 B equipped with a high-intensity xenon flash lamp. A Branson 1210 sonicator was used to help dissolve the osmium complex in solvent when needed. Thermo Scientific digital pipets were used for the addition of all solutions into the quartz cuvette.
The stock solutions of the osmium complexes were prepared by dissolving 0.6–2.8 mg of each osmium complex in ethanol (ethanol solution) or distilled water (aqueous solution) in a 100 mL volumetric flask. The 1.0 mg/mL heparin aqueous solution was prepared by dissolving 5.0 mg of heparin in 5.00 mL of distilled water. The 0.10 mg/mL and 0.010 mg/mL heparin aqueous solutions were diluted from the 1.0 mg/mL stock solution. For small anions solutions, a 0.100 M solution of each salts was first made and then diluted to 0.0100 M and 0.00100 M.
For the luminescence measurements of each osmium complex solution (ethanol, aqueous), 2 mL stock solution was transferred to a quartz cuvette (1 cm × 1 cm × 4.4 cm) placed in the luminescent spectrometer cuvette holder. The emission and excitation slits were both set at 10 nm for the luminescence measurements. A prescan was taken first to obtain the wavelengths of the maximum excitation and emission intensities; then the emission spectrum was recorded by exciting the sample at the appropriate excitation wavelength. In order to study the anion quenching or enhancement of the luminescence of each osmium complex solution, 2.00 mL of the stock solution was placed in the cuvette, and the emission spectrum was measured; then a small amount (10
Detection of injectable heparin was performed using standard addition method. First, 10 or 20
The excitation and emission spectra of each complex in their ethanol solution (except for [Os(bpy)2CO(py)](PF6)2 which could not dissolve in ethanol) and their aqueous solution were first recorded at room temperature under ambient air. All the spectra exhibited broad emission and excitation bands up to 200 nm in width. Figure
Luminescence spectra of (a) [Os(phen)2CO(PPh3)](PF6)2 and (b) [Os(phen)2CO(py)](PF6)2 in ethanol solution (2.4 mg/100 mL).
As shown in Table
Excitation and emission maxima wavelengths (nm) of the six osmium complexes.
Complex |
|
|
|
|
---|---|---|---|---|
|
520 | 520 | 396 | 396 |
|
520 | 520 | 396 | 396 |
|
560 | 560 | 420 | 420 |
|
560 | 560 | 430 | 430 |
|
560 | 560 | 420 | 420 |
|
N/A | 560 | N/A | 420 |
The spectral changes that occur before and after the addition of small inorganic anion solutions suggest that those anions
It was expected that heparin would quench the luminescence of the osmium complexes due to its high negative charges. Surprisingly, however, it was found that heparin actually enhanced the luminescence intensity of the osmium complexes in ethanol solutions. The luminescence of each complex in the ethanol and aqueous solution changed quite differently after heparin was added.
In ethanol solution, each complex exhibited an increase in luminescence intensity after heparin was added in concentrations greater than 0.5
Figure
Luminescence spectra of [Os(bpy)2CO(4-phpy)](PF6)2 in ethanol solution (2.8 mg/100 mL) in the presence of varing concentrations of heparin. From bottom to top, heparin concentration: 0, 9.9, 19.6, 29.1, and 38.5
Plot of the luminescence enhancement ratio (
The heparin enhancement of the luminescence of [Os(phen)2CO(py)](PF6)2 (2.4 mg/100 mL EtOH) is smaller than that seen for the 4-phpy complexes. The luminescence intensity was enhanced by two and half times when the added heparin concentration was increased to 20–30
The heparin enhancement of the luminescence of the [Os(phen)2CO(PPh3)](PF6)2 (2.4 mg/100 mL EtOH) and the [Os(bpy)2CO(PPh3)](PF6)2 (2.1 mg/100 mL EtOH) solutions is very small compared to the Os-(4-phpy) and Os-py complexes. Here, the shift in the emission maxima is insignificant (less than 2 nm).
Figure
Plot of the luminescence enhancement ratio (
The osmium complexes luminescence enhancement by heparin is likely contributed by the close contact of heparin and the complexes, which forms a rigid structure that reduced the solvent collision and quenching. Heparin has a long linear structure with anionic sites on its branches when it is dissolved in aqueous solution [
Schematic drawing of the interaction of heparin with the double positive-charged osmium complexes.
The sixth ligand in these complexes plays an important role in the interaction of these charged sites. The complexes [Os(bpy)2CO(4-phpy)]2+, [Os(phen)2CO(4-phpy)]2+, or [Os(phen)2CO(py)]2+ have small narrow 4-phenylpyridine or pyridine ligand. When heparin is added to their ethanol solution, these osmium complexes could readily approach the heparin polyanion and bind up to two heparin anion sites. These two sites may well form two heparin polyanions branches, thus forming ion pairs between the polyanion and osmium complex. Therefore, heparin can cause significant enhancement to the luminescence of the Os-(4-phpy) and Os-py complexes. Further, the luminescence only stops increasing at very high heparin concentrations when all the osmium complex cations are bound to heparin anion sites. By contrast, the [Os(phen)2CO(PPh3)]2+ and [Os(bpy)2CO(PPh3)]2+ complexes have a sterically bulky PPh3 group, which prevents them from approaching the heparin polyanion closely. Therefore, the luminescence intensity of the Os-PPh3 complexes could not be significantly enhanced by heparin.
The effect of heparin on the emission of these osmium complexes in their aqueous solution is quite different from that of their ethanol solution. The aqueous solution of osmium complexes with a PPh3 or pyridine group exhibits a decrease in luminescence intensity at low added heparin concentrations; then the luminescence increases at heparin concentration above 15 to 19
Figure
Plots of the luminescence enhancement ratio (
In the aqueous solutions of [Os(phen)2CO(PPh3)](PF6)2 or [Os(bpy)2CO(PPh3)](PF6)2, it is even harder for the osmium complex to bind to heparin because of the high solubility of heparin in aqueous solution and the hindrance of the bulky PPh3 group. In fact, at low heparin concentration, heparin quenches the luminescence of these two complexes heavily as polyanion. The decrease of the luminescence intensity in the aqueous solution of these two complexes fits the Stern-Volmer equation
Here
Stern-Volmer plot is shown in Figure
Stern-Volmer plot for the heparin quenching of the luminescence of [Os(phen)2CO(PPh3)](PF6)2 aqueous solution (1.2 mg/100 mL in H2O).
Table
Linear response ranges and slopes obtained from the
Complex | Solution | Linear range ( |
Slope |
---|---|---|---|
|
Ethanol solution | 1–38 | 0.061 |
|
Ethanol solution | 1–40 | 0.072 |
|
Ethanol solution | 1–24 | 0.074 |
|
Aqueous solution | 1–10 | 0.116 |
|
Aqueous solution | 1–10 | −0.036* |
The detection of heparin was then performed with commercial medicinal solutions to evaluate the feasibility. Two commercial heparin sodium injection solutions (40 and 100 units/mL in 5% dextrose, in 100 mL plastic bags) from B. Braun Medical Inc. were tested. With the [Os(phen)2CO(4-phpy)]2+ complex in aqueous solution, the emission intensities of the osmium solution before and after adding 20
To eliminate the background effect, the standard addition method was used. First, 10 or 20
The luminescence of osmium carbonyl complexes is stable at room temperature in ethanol and aqueous solutions, though the intensity and wavelength of this emission are heavily dependent on the other five ligands. The effect of heparin on the luminescence of the osmium complexes depends heavily on both the ligand and solvent used. In ethanol solutions, heparin could enhance the luminescence of five osmium complexes at low heparin concentrations, with the greatest enhancement occurring with the Os-(4-phpy) complexes. This increased emission intensity may be due to binding of heparin to the double charged complexes which changed the solvent environment of the complexes. In aqueous solutions, heparin quenching the osmium complex emission was more significant as heparin is more soluble in water and behaves as an anion quencher. These osmium complexes are shown to be useful for the fast and sensitive detection of heparin in commercially injectable samples.
The authors are thankful to St. John’s University for the financial support throughout this research.