molecularly imprinted polymers ( mips ) exhibiting high selectivity and affinity to the target molecule are well recognized as a fast growing research field [ 1 , 2 ] . they have been successfully applied in various novel methods of chemical analysis [ 3 , 4 ] , including potentiometric sensors [ 57 ] , amperometric detection , and differential pulse cathodic stripping voltammetry . an optical sensor was fabricated with an mip film for the determination of formaldehyde molecules that induced measurable optical reflectivity shifts . for surface plasmon resonance spectroscopy , mip particles were spin - coated onto a gold surface to detect theophylline . a novel sensing scheme based on nonlinear fluorescence quenching of 17-estradiol ( e2 ) was recently developed in our laboratory . small nitrite ions penetrated the porous structure of mip particles and quenched the fluorescence emission from e2 molecules inside imprinted cavities . on the contrary , large methacrylamide molecules ( 3-d stearic diameter = 0.536  nm ) were hindered when penetrating the pores to access the imprinted cavities , resulting in low dynamic quenching . research was continued in our laboratory to evaluate larger quenchers , such as gold nanoparticles ( aunps ) that could readily be synthesized with a diameter of 43    5  nm [ 1416 ] . their effectiveness was studied with regard to quenching the fluorescence of only nonspecifically bound e2 molecules throughout the porous mip particles , but not those specifically bound inside the imprinted cavities , as illustrated in scheme 1 . the objective of this work was to develop a rapid method ( hopefully under 5  min ) for the determination of trace e2 in water ( down to 0.1  ppb ) . sodium citrate tribasic dihydrate , gold ( iii ) chloride trihydrate , sodium nitrite , and e2 were purchased from sigma - aldrich ( st . louis , mo , usa ) . methacrylic acid and methacrylamide were purchased from aldrich ( milwaukee , wi , usa ) . 2,2-azobisisobutyro - nitrile ( aibn ) was purchased from pfaltz and bauer ( waterbury , ct , usa ) . methanol ( hplc grade ) , acetonitrile ( hplc grade ) , and acetone ( spectro grade ) were purchased from caledon ( georgetown , on , canada ) . acetic acid ( reagent grade ) 18-mcm distilled deionized water ( ddw ) was obtained from a millipore milli - q water system ( bedford , md , usa ) . these particles ( 80  ml ) were washed with 15% acetic acid in methanol ( v / v ) , methanol , acetonitrile , and ddw three times . each washing was combined with 1  hr of sonication and 1  hr of centrifugation to completely extract template e2 molecules from the particles and remove polymerization reagent residues . after the last washing with ddw , the ph was 5.5    0.1 in the supernatant and the free e2 concentration was below the detection limit of hplc - fd instrument . another batch of freshly prepared mip submicron particles was washed only with ddw for 25 times . the aunps , mip , and e2-mip particles were suspended in 10  m kno3 at a concentration of 40  mg / ml . the suspensions were sonicated for 15  min before measurement on a nanodls particle size analyzer ( brookhaven instruments , holtsville , ny , usa ) . the instrument had been calibrated by 92    4  nm nanosphere size standards ( duke scientific , palo alto , ca , usa ) . a total of 10 measurements were run after 30  s of quiescence time , and the laser beam intensity was automatically optimized by the analyzer before each run .   3.5  ppm e2 ( 2  ml ) and 2.5  mg / ml e2-mip particles ( 2  ml ) were added into two 3-ml quartz cuvette cells , each equipped with a polytetrafluoroethylene ( ptfe ) stopper . then 1  ml of aunps aqueous suspension with elemental concentrations from 0  m to 5.88  10  m was used to quench the e2 and e2-mip particles fluorescence emission intensities . all emission intensities were measured at room temperature ( 20    1c ) by a fluorescence spectrophotometer ( varian cary eclipse , palo alto , ca , usa ) using excitation wavelength of 280    1  nm and emission wavelength of 310    1  nm ( or scanning from 290  nm to 450  nm ) . both the excitation and emission slits were set at 5  nm . to test for inner filter effect , 1  ml of 1% ( w / w ) trisodium citrate dihydrate was used instead of aunps . similarly , e2 and e2-mips fluorescence quenching experiments with sodium nitrite were accomplished under exactly the same experimental conditions . 7.7    0.1  mg , 5.5    0.1  mg , 3.3    0.1  mg , and 1.1    0.1  mg of template - removed mip submicron particles were added into 2.2  ml of e2 aqueous solution with concentrations from 0.0001  ppm to 3.5  ppm . the blank and e2-templated mip particles were prepared by using the same amount of template - removed mips and e2-mip particles suspended in 2.2  ml of ddw . all of these suspensions were incubated under sonication for 535  min at room temperature ( 20    1c ) . then , 2.0  ml of e2-bound mip or e2-mip submicron particle suspension was transferred into a 3  ml quartz cuvette cell and spiked with 1  ml of 5.88  10  m aunps aqueous suspension . after the fluorescence emission intensity was recorded , 100  l of 150    1  mg / ml sodium nitrite was added to perform the second step of fluorescence quenching . all light absorption spectra by quenchers were measured on a uv - visible spectrophotometer ( varian cary 3 , palo alto , ca , usa ) by scanning from 250  nm to 350  nm at room temperature ( 20    1c ) to investigate the inner filter effect . the absorption of both exciting light (  
 ex = 280  1  nm ) and fluorescence emission (  
 em = 310  1  nm ) by quenchers was corrected , for right - angle illumination , as described elsewhere . after it interacts with nonfluorescent mip particles both specifically and nonspecifically , the bound e2 molecules can be determined by spectrofluorimetry (  
 ex = 280  nm and  
 em = 310  nm ) . figure 1 shows the fluorescence emission spectra of e2 , e2-mip particles , and e2-bound nip particles during their quenching by aunps . without particles , a 3.5  ppm e2 aqueous solution exhibited decreasing fluorescence intensities when aunps were added stepwise as shown in figure 1(a ) . the fluorescence intensity decreased by almost 82% from the initial level as the concentration of aunps reached 5.88  10  m. similarly , the quenching effects of aunps on e2-mip particles and e2-bound nip particles are evidenced in figures 1(b ) and 1(c ) , decreasing the fluorescence intensity by 76% and 77% . the strong mie scattering peak ( at 280  nm ) from particles slightly enhanced all e2 fluorescence emission peaks at 310  nm , which can be considered meritorious for the determination of e2 at trace levels . two raman scattering peaks ( at 380  nm and 425  nm ) were characteristic of particles when an excitation wavelength of 280  nm was used . luckily , they did not have any significant impact on the fluorescence quenching results . the fluorescence properties of aunps were studied before they were used as a large fluorescence quencher in all subsequent experiments . when 5.88  10  m of aunps were examined by scanning the excitation wavelength in figure 2(a ) and using an emission wavelength of 310  nm , only one mie scattering peak was observed at 310  nm . when the emission wavelength was scanned in figure 2(b ) using an excitation wavelength of 280  nm , only two mie scattering peaks were found at 280  nm ( first order ) and 570  nm ( second order ) . therefore the aunps were nonfluorescent , making them ideal for use as fluorescence quencher in this work . figure 2(c ) shows the fluorescence emission spectrum of 3.5  ppm e2 aqueous solution while figure 2(d ) shows the same spectrum after addition of trisodium citrate dehydrate ( 1%  w / w ) . no significant inner filter effect was observed from 1% trisodium citrate dihydrate , which was present in the synthesis of aunps . furthermore in real samples , e2 may exist with some metabolites or other related compounds that can fluorescence . however , no interferences would be possible because these other fluorescent compounds can not bind with the mip cavities . therefore after fluorescence quenching by aunps , the interferences can be eliminated . in our previous study , sodium nitrite was able to quench the fluorescence emissions from e2 aqueous solution , e2-mip aqueous suspension , and e2-bound nip aqueous suspension . hence , it was used in this work to finish the fluorescence quenching job after aunps quenched only the fluorescence emission from e2 that were nonspecifically bound inside particles . as shown in figure 3 , the residual fluorescence emission from 2.5  mg / ml e2-mip particles in aqueous suspension , after quenching with 5.88  10  m aunps , was an intensity of 14.2    0.2 arbitrary units ( a.u . ) coming mostly from e2 specifically bound inside the mip cavities . sodium nitrite was then titrated , stepwise from 0  m to 6.5  10  m , into the mixture of e2-mip particles and aunps . due to its small size , the nitrite anion penetrated through the porous mip particles and quenched the fluorescence emission from the specifically bound e2 molecules . at the end of titration , the emission intensity was reduced to 5.5    0.2  a.u . this result clearly demonstrated how simple it was to determine the amount of specifically bound e2 molecules . figure 4 plots all the fluorescence emission intensities measured ( at 310  nm ) from figures 1 and 3 . intuitively , both free e2 molecules in aqueous solution and nonspecifically bound e2 molecules in nip particles ( which had no imprinted cavities ) in aqueous suspension were all accessible by aunps to undergo collisional quenching . if there were no imprinted cavities in mip particles to protect the specifically bound e2 molecules ( from quenching by aunps ) , the final emission intensity in figure 4(a ) would probably be as low as the ~10  a.u . in figures 4(b ) and 4(c ) plots when the aunps quencher concentration reached 38.7  ppm (= 5.88  10  m. in reality , the e2-mip particles contained some inaccessible e2 molecules that contributed to a higher final emission intensity of ~18  a.u . the mip and nip particles studied in this work had diameters , as measured by a nanodls particle size analyzer , of 477    11  nm and 373    21  nm , respectively . e2 molecules were specifically bound inside the mip cavities that were complementary in size , shape , and arrangement of functional groups . small nitrite anions could easily penetrate the porous mip particles and quench the fluorescence from the e2 molecules by dynamic collisions . the large aunps used in this study had a diameter of 43    5  nm . figure 5 shows the correlation of light scatting intensity with time , as obtained for aunps during a particle size measurement . over 10 runs , the particle size readings varied between 33  nm and 85  nm with a polydispersity of 0.3 ( moderate dispersion ) . the size range seemed to be suited for fluorescence quenching of e2 molecules that were nonspecifically bound to mip particles . figure 6 plots the fluorescence quenching efficiency (  = 1  f / f 
 0 , where f and f 
 0 are the fluorescence emission intensities measured in the presence and absence of quencher ) versus the concentration of quencher . with aunps , similar quenching efficiencies were observed for both particles and e2 in ( a ) , ( b ) , and ( c ) . by comparison , sodium nitrite exhibited significantly lower quenching efficiency in ( d ) and ( e ) . approximately 4500  ppm sodium nitrite was needed in ( e ) to quench 80% of fluorescence emission from e2-mip particles although only 38.7  ppm aunps was needed in ( b ) . interestingly a lesser amount of sodium nitrite was needed in the presence of aunps in ( d ) to quench e2-mip particles , from 80% to 90% , than in ( e ) . all fluorescence quenching data were analyzed further by applying the stern - volmer ( s - v ) equations that examine different quenching mechanisms [ 18 , 22 ] : 
 ( 1)f0f=1+ksv[q ] , 
 ( 2)f0f=(1+ksv[q])exp  ( v[q ] ) . 
 f 
 0 and f are the fluorescence emission intensities in the absence and presence of quencher . k 
 sv denotes the dynamic quenching constant , and v denotes the static quenching constant . [ q ] is the concentration of quencher . equation ( 1 ) represents a linear function between dynamic quenching and quencher concentration , where quencher collision with the excited fluorophore ( e2 * ) returns it to the ground state without fluorescence emission .   figure 7 shows the linear s - v plots for aunps , which were best analyzed using ( 1 ) . the k 
 sv for e2 is 2.6 ( 0.1 )  10  m ( r 
  = 0.9478 ) , the k 
 sv for e2-mip particles is 2.9 ( 0.1 )  10  m ( r 
  = 0.9566 ) , and the k 
 sv for e2-bound nip particles is 3.3 ( 0.1 )  10  m ( r 
  = 0.9678 ) . due to their large size , aunps could hardly penetrate the porous structures of e2-mip and e2-nip particles . consequently , their k 
 sv values were in the same order of magnitude as that obtained for e2 in aqueous solution . the s - v plots for sodium nitrite was found to be nonlinear ( with an upward - curving trend ) , as shown in figure 8 . nonlinear s - v plots had been discussed by zhao and swager   as a combined result from dynamic and static quenchings . in contrast to dynamic quenching , the mechanism of static quenching involves interaction between the quencher and fluorophore to form a nonfluorescent complex . analysis using ( 2 ) obtained k 
 sv = 2.1  10  m and static quenching constant ( v ) below 1.0  m ( r 
  = 0.9220 ) for 2.5  mg / ml e2-mips in aqueous suspension containing 5.88  10  m aunps . since mip cavities did not facilitate complex formation between e2 molecules and nitrite anions due to space constraints , the v value turned out to be very small . a higher v value of 4.7  m was obtained for 4.5  ppm e2 in aqueous solution , which signifies the complexation of e2 molecules with nitrite anions in the absence of steric hindrance . various concentrations ( from 0.1  ppb up to 3.5  ppm ) of e2 in aqueous solution were used to validate mip particles for rapid e2 determination by fluorescence quenching . mip particles were added in these e2 solutions to form 0.53.5  mg / ml suspensions . two incubation times ( 5  min and 35  min ) were tested to investigate binding equilibrium between e2 molecules and mip particles . the fluorescence emission intensity from e2 specifically bound with mip particles was determined by measuring the fluorescence emission intensities after two quenching steps , as summarized by 
 ( 3)ie2  inside  mip  cavities = iafter  quenching  with  aunps    iafter  quenching  with  sodium  nitrite . essentially , the first quenching with aunps would eliminate the fluorescence emission from e2 molecules on nonspecific binding sites throughout the porous mip particles . then , sodium nitrite would quench the fluorescence emission from e2 molecules inside the specific imprinted cavities . the normalized % binding of e2 with imprinted cavities was determined as 
 ( 4)normalized  %  e2  binding=  iimax  , 
 where i is the fluorescence intensity from e2 bound specifically inside imprinted cavities for an e2 standard solution , and i 
 max is the maximum fluorescence intensity from e2 bound specifically inside all imprinted cavities . both i and i 
 max were calculated according to ( 3 ) , in parallel measurements . to determine i 
 max , e2-mip particles were prepared by washing only with ddw to remove all nonspecifically bound e2 molecules . normalization was deemed necessary because i 
 max is not linearly dependent on the concentration of e2-mip particles in aqueous suspension , as shown in figure 9 , due to inner filter effects . figure 10 shows % e2 binding ( with imprinted cavities ) as a function of e2 concentration after incubation for ( a ) 5  min and ( b ) 35  min . a comparison of ( a ) with ( b ) indicates that % e2 binding exhibited no significant difference . this suggests that binding equilibrium was reached in 5  min or less , in agreement with the ~2  min previously reported . as 0.5  mg / ml e2-mip particles had the smallest number of imprinted cavities and hence the lowest i 
 max value as the method involved binding of the analyte with mip particles for the best possible selectivity ( only second to natural antibodies ) , scatchard plots were constructed in accordance with the following equation : 
 ( 5)%  e  binding=  [ e2](kd+[e2 ] ) , 
 where k 
 d is the equilibrium binding constant . the best calibration curves , fitted using graphpad prism , are shown as solid lines in figure 10 . the best correlation coefficients were obtained for 2.5  mg / ml mip particles , being r 
  = 0.9716 for 5  min of incubation and 0.9937 for 35  min of incubation . table 1 shows that the equilibrium binding constant ( k 
 d ) for 2.5  mg / ml of mip particles , using 5  min of incubation , was the highest among the three mip concentrations studied as expected . aunps ( 43    5  nm ) were efficient in quenching the fluorescence emission from e2 molecules in aqueous solution , or nonspecifically bound with porous particles . for e2 molecules specifically bound inside imprinted cavities , a rapid method ( under 10  min ) has been developed for e2 determination by measuring the change in fluorescence emission intensities between these two fluorescence quenching steps , using first aunps and then sodium nitrite . one major advantage of this method is the high selectivity of mip particles for e2 , as previously demonstrated using molecules with similar structures ( estrone , ethynylestradiol )   and dissimilar structures ( bisphenol a ) . other fluorescent molecules would not interfere with the e2 determination because they can only bind nonspecifically to be readily quenched by aunps . highly correlated scatchard plots ( r 
  > 0.97 ) serve well as a standard calibration curve . the detection limit for e2 is low , at the ultratrace level of 0.1  ppb . further work is underway to verify that no potential interference by common organics and anions ( co3 
 , no3 
 , ptcl4 
 , scn , n3 
  present in environmental waters ) exists after an extra centrifugation step is added ( to precipitate the mip particles out for transfer into a cuvette of deionized water ) before the two fluorescence quenching measurements . optimization of the method will also be completed with testing of real sample matrices from environmental waters .
a new method was optimized for rapid screening of 17-estradiol ( e2 ) in water under 10  min . 
 molecularly imprinted polymer ( mip ) particles ( 325    25  nm ) were added in a water sample at ph 5.5 and 20c to form a suspension . 
 fluorescence emission from e2 nonspecifically bound onto the mip particles was first quenched by large gold nanoparticles ( 43    5  nm ) . 
 the stern - volmer plot was linear , with dynamic quenching constants ( k 
 sv ) of 2.9 104  m1 . 
 fluorescence emission from e2 specifically bound inside the mip particles was next quenched by small nitrite anions that easily penetrated the imprinted cavities . 
 the stern - volmer plot became nonlinear , with k 
 sv = 2.1  102  m1 and static quenching constant ( v ) below 1.0  m1 . 
 the difference between these two emission intensities varied as the initial e2 concentration in water , generating a scatchard calibration curve with r 
 2 > 0.97 from 0.1 to 10  ppb .