heparina , a highly sulfated glycosaminoglycan , has the highest negative charge density of any known natural biological molecule .
heparin has been extensively used as an injectable anticoagulant in many clinical procedures for the prevention of blood clotting , especially during open heart surgery [ 2 , 3 ] .
pharmaceutical - grade heparin is derived from mucosal tissues of slaughtered meat animals such as porcine intestine and bovine lung .
native heparin is a polymer with a molecular weight ranging from 3,000 to 50,000 , and the average molecular weight of most commercially prepared heparin is in the range of 12,000 to 15,000 . under physiological conditions ,
the sulfate groups are deprotonated and attract positively charged counterions ( usually sodium ) to form a heparin salt .
it is in this form that heparin is usually administered as an anticoagulant [ 5 , 6 ] . at the end of the medical procedure , the heparin has to be rendered inactive to prevent possible bleeding problems .
therefore , clinicians need to know the exact concentration of heparin remaining when they attempt to neutralize the anticoagulant activity of heparin via precise doses of protamine . the current heparin detection method used in hospital
is based on the blood clotting time [ 7 , 8 ] and varies widely due to variation in the blood content of the different individuals .
thus , the discovery of accurate , rapid , reliable safe , and low - cost methods of heparin detection remains an important area of sensor research .
for example , electrochemical sensors [ 9 , 10 ] and optical sensors [ 11 , 12 ] were intensively studies in the 1990s , and more recent studies have focused on fluorescent spectroscopy [ 13 , 14 ] and uv / vis spectroscopy using gold nanoparticles .  
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 .
however , the luminescence intensity of the chromophore ( or excited state complex ) can be seriously reduced by collision with other chemical species or enhanced by chemical interactions . through studying the effect of these chemical species on the luminescence intensity of the chromophore ,
the quantitative determinations of a variety of important inorganic and organic species in trace amounts have been realized by photoluminescence methods .  
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 [ 17 , 18 ] .
this research focuses on studying the effect of the heparin polyanion on the photoluminescence of these polypyridyl osmium(ii ) complexes , for the purpose of finding potential luminescence markers for the detection of the optically nonactive polyanion heparin . to our surprise
, polyanions show unique influences on the luminescence spectra and intensity of these osmium complexes .
the 1,10-phenanthroline ( phen ) complexes were compared with the 2,2-bipyridine ( bpy ) complexes .
for each of these chromophoric ligands , three analogous sets were compared using pyridine ( py ) , triphenylphosphine ( pph3 ) , or 4-phenylpyridine ( 4-phpy ) as the sixth ligand .
these ligands showed a significant impact on the luminescence characteristics of the osmium complex in the absence as well as in the presence of polyanions .
the results of the studies could lead to a new , potential effective detection method for heparin monitoring .
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 1 . the synthesis of six osmium complexes was described in [ 17 , 18 ] .
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 .
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 0.62.8  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 
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  l or 20  l ) of the anion solution ( 0.010~1.0  mg / ml for heparin and 0.10 to 0.0010  m for small anions ) was added to the cuvette and the emission spectrum was recorded after the solution was well mixed .
the anion solution was continuously added to the cuvette , and the emission spectrum was recorded after each addition .
the values of the luminescence intensity were taken at a fixed wavelength at or near the emission maximum .
first , 10 or 20  l of injectable heparin solution was added to 2  ml of the aqueous [ os(phen)2co(4-phpy)](pf6)2 solution .
next three portions of 5  l of 1  mg / ml standard heparin solution were added , and the emission spectrum of the solution was measured before and after each addition .
the concentrations of the heparin in the injectable solution were calculated using the standard addition curve .
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 2 shows the emission spectra in ethanol solution of the two complexes : [ os(phen)2co(pph3)](pf6)2 , and [ os(phen)2co(py)](pf6)2 , which exibit the largest difference in luminescence intensity .
as shown in table 1 and figure 2 , the two os - pph3 complexes have much higher luminescence intensities and shorter emission wavelengths than the two os-(4-phpy ) complexes and the two os - py complexes ( 520  nm versus 560  nm ) . the shorter wavelength ( higher energy ) of the emission
is caused by the -backbonding ability of the phosphine which stabilizes the homo of the molecule relative to pyridine .
the higher luminescence intensities are attributed to the larger steric effect of the pph3 ligand which shields the complex from colliding with the solvent .
this slows down the vibrational relaxation to the ground vibrational level of the excited state that must occur prior to emission of a photon of light that occurs when the electron returns to the ground electronic state .
all these six complexes exhibit higher luminescence intensities in aqueous solution than in ethanol solution due to the hydrophobicity of the ligands surrounding the osmium center .  
the spectral changes that occur before and after the addition of small inorganic anion solutions suggest that those anions clo4 
 , so4 
 , ch3coo , cl , br and no3 
  exhibit very little quenching of the luminescence of the osmium complex with anion concentrations up to 0.01  m , while no2 
 , scn and i anions can dramatically quench the luminescence of the osmium complexes with concentrations at 0.01  mm .
however , oh exhibits a small enhancement of the luminescence of the osmium complex ; luminescence intensity slightly increased as the solution ph increased from 6 to 9 in aqueous solution .  
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  g / ml . however , the amount of increase varied significantly for each complex . the solutions of [ os(bpy)2co(4-phpy)](pf6)2 , [ os(phen)2co(4-phpy)](pf6)2 , and [ os(phen)2co(py)](pf6)2 exhibited a very large increase in luminescence intensity while the solutions of [ os(phen)2co(pph3)](pf6)2 and [ os(bpy)2co(pph3)](pf6)2 exhibited a small luminescence increase .  

figure 3 shows the luminescence spectra of the ethanol solution of [ os(bpy)2co(4-phpy)](pf6)2 in the absence and presence of heparin at different concentrations .
the luminescence intensity was increased from a relative emission intensity of 136 in the absence of heparin to 462 by addition of heparin in a concentration range of 5.038.5  g / ml .
further , there is an 8  nm blue shift in wavelength of the emission maxima with high heparin concentrations . a plot showing the wider range of heparin concentrations on the enhancement ratio ( f / f 
 0 ) is shown in figure 4 .
here , f and f 
 0 are the luminescence ( fluorescence ) intensity of the osmium complexes solutions at 560  nm with and without heparin , respectively .
the luminescence intensity increased steadily up to a heparin concentration of 56  g / ml . in the lower concentration range ( up to 30  g / ml ) , there is a linear relationship between the f / f 
 0 ratio and the concentration with an r 
  of 0.99 .
the heparin enhancement of the [ os(phen)2co(4-phpy)](pf6)2 ( 2.4  mg/100  ml etoh ) luminescence has a similar pattern to that seen with [ os(bpy)2co(4-phpy)](pf6)2 . in this case , the luminescence was enhanced by three and half times when the added heparin concentration was increased to 50  g / ml . once again , there is an 8  nm blue shift of the emission maxima at higher heparin concentrations . in the concentration range of 1 to 40  g
/ ml , there is a linear relationship between the f / f 
 0 ratio and the heparin concentration with an r 
  of 0.99 .  
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 2030  g / ml , and then the luminescence started to decrease at heparin concentrations in excess of 30  m / ml . in this case , there is also an 8  nm blue shift of the emission maxima at higher heparin concentrations .  
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 5 shows the plot of f / f 
 0 versus heparin concentration for the two phenanthroline complexes with pyridine and pph3 as the sixth ligand . from figures 4 and 5
, it can be seen that the 4-phpy complexes , which have the lowest solution luminescence in the absence of polyanions , showed the highest enhancement in the concentration range of 540  g / ml added heparin concentrations . by contrast , the pph3 complexes showed the smallest enhancement in the same concentration range .  
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 .
when its aqueous solution is added to the ethanol solution of the osmium complex , the anion sites on its branches attract the double positively charged osmium complex cations . as a result , the osmium complex could electrostatically bind to heparin and become surrounded by the heparin molecules ( figure 6 ) . in this case , solvent molecules can no longer get close enough to the osmium complex to help relax the excited state , resulting in the emission maximum moving to higher energy .
this effect would also explain the increased emission intensity since the rate of nonradiative decay would be greatly reduced in the absence of favorable interaction with solvent molecules .
the sixth ligand in these complexes plays an important role in the interaction of these charged sites .
the complexes [ os(bpy)2co(4-phpy ) ] , [ os(phen)2co(4-phpy ) ] , or [ os(phen)2co(py ) ] 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 ) ] and [ os(bpy)2co(pph3 ) ] 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  g / ml . however , complexes with the 4-phpy group exhibit a decrease of luminescence at heparin concentrations below 0.5  g / ml then an increase in luminescence intensity is observed with the addition of heparin in the range of 1 to 15  g / ml .
this shows that as a polyanion , when not binding to the osmium complexes , heparin is an efficient luminescence quencher in aqueous solutions .
this is especially true with the pph3 complexes , where binding only occurs at very high ( > 15  g / ml ) heparin concentrations . by contrast , the 4-phpy complexes bind more strongly to heparin , and so less of a quenching effect is observed . 

figure 7 shows the plots of the luminescence enhancement ratio ( f / f 
 0 ) as a function of heparin concentration for the two os-(4-phpy ) complexes in aqueous solution .
the high luminescence enhancement ratio of the 1,10-phenanthroline complexes by heparin shown in figure 7 suggests that the [ os(phen)2co(4-phpy ) ] complex binds more strongly to the heparin polyanion than the analogous 2,2-bipyridine complex complexes . comparing figures 4 , 5 , and 7 ,
the enhancement of heparin to the [ os - co(bpy)2(4-phpy ) ] complex is much smaller in aqueous solutions , which indicate a weaker binding to heparin , possibly due to the higher solubility of heparin and the complexes in aqueous solution .   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 
 ( 1)f0f=1+ksv[q ] . 
  here f 
 0 and f are the luminescence signals in the absence and in the presence of a quencher , respectively ; k 
 sv is the stern - volmer quenching constant ; and [ q ] is the concentration of the quencher .   stern - volmer plot is shown in figure 8 for the [ os(phen)2co(pph3 ) ] complex .
the high linearity of the stern - volmer curve , shown at the concentration range of 1 to 12  g / ml in the insert , confirms that heparin exhibits dynamic quenching to the os - pph3 complexes .
the nonlinearity at higher concentrations may indicate that osmium complex - heparin ion - pairs have formed .
further addition of heparin above 12  g / ml caused the luminescence of these complexes to increase slightly . 

table 2 summarizes the heparin concentration ranges that induced significant luminescence changes to the complexes studied in ethanol or aqueous solutions . in ethanol solution ,
[ os(bpy)2co(4-phpy)](pf6)2 exhibits the greatest enhancement in the presence of heparin as measured by the highest slope of the f / f 
 0 versus heparin concentration linear curve .
this complex exhibits the longest response range and the highest sensitivity to heparin concentration . in aqueous solutions ,
the [ os(phen)2co(4-phpy)](pf6)2 and both phosphine complexes showed significant luminescence changes at low heparin concentrations .  
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 ) ] complex in aqueous solution , the emission intensities of the osmium solution before and after adding 20  l of these two heparin solutions were measured .
the ratios ( f / f 
 0 ) obtained were 1.30 and 1.97 ( n = 5 ) , respectively , showing that the osmium complex indeed had a greater response to higher heparin concentration preparations .
however , these medical preparation responses are much higher than the pure heparin solution response .
when response curve of heparin in the presence of 5% dextrose was performed , an enhanced osmium complex response to heparin concentration was observed .
this enhanced response is likely due to the hydrophobic environment that the polysaccharide , dextrose , created around the osmium complex in aqueous solution , thus reducing the extent of nonradiative decay of the excited states and increasing the luminescence intensity of the osmium complex .   to eliminate the background effect ,
first , 10 or 20  l of the commercial heparin sample solutions was added to the aqueous osmium complex solution ; then three portions of 5  l of the 1.0  mg / ml heparin standard solution were added .
the luminescence values of the complex solution after each addition of the sample and standard heparin solutions were measured at 560  nm . with the standard addition curves and using the conversion of 180  units / mg of sodium heparin ,
the average heparin concentration in the two sample bags was calculated to be 46  3 and 111 
the relative standard deviation , rsd for the two corresponding samples are 5.6 ( n = 3 ) and 2.3 ( n = 6 ) respectively , indicating that the precision of the method is acceptable considering the volume added is very small , although they are 11% higher than the labeled value . further tests , with other commercial sources having different backgrounds , are currently under study .
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 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 ( f / f 
 0 ) versus the heparin concentration range of 140  g / ml . in aqueous solutions , heparin quenching of the complexes was more significant ; a linear quenching curve was obtained with [ os(phen)2co(pph3)](pf6)2 in the lower concentration range of 112  g / ml . the interaction of these complexes with heparin in the solutions is discussed . 
 the complexes are shown to be successful in the fast and sensitive detection of heparin in commercial injectable samples . 
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion