semiconductor quantum dots are excellent fluorescent nanoprobes for biological and biomedical applications because of their unique size - dependent optical and electronic properties ( penn et al 2003 ; alivisatos
these nanoparticles ( nps ) possess unique optical properties in comparison with traditional organic dyes including size- and composition - tunable emission , narrow emission spectra , and excellent photostability ( hotz 2005 ) .
recent improvements in surface chemistry have augmented the ability to make functionalized nps to create bioconjugated nps that allow for specific targeting or signal enhancement ( farokhzad et al 2006 ; fujiwara et al 2006 ; goodman et al 2006 ; ipe et al 2006 ; suk et al 2006 ) .
bioconjugated nps , coupled with spectroscopy or other spectral imaging tools , eg , flow cytometry and histological techniques , provides a novel and powerful platform for mapping the molecular and disease profiles associated with infection by different pathogens ( zhao et al 2004 ; driskell et al 2005 ; yao et al 2006 ; mcveigh 2006 ) .
another advantage of bioconjugated nps over conventional pathogen detection methods is that the nps may be conjugated with many biomolecules thereby enhancing the signal and the binding avidity by mitigating a multivalency effect .
this is particularly important for targeting nps and for increasing the delivery or efficacy of bioconjugated molecules .
however , only limited reports are available which address the utility of nps for detection or diagnosis virus infection ( agrawal et al 2005 , 2006 ; bentzen et al 2005 ; driskell et al 2005 ; li , cu , et al 2005 ; li , liu , et al 2005 ; wabuyele and vo - dinh 2005 ; wan et al 2005 ; fuentes et al 2006 ; liu , cao , et al 2006 ; souza et al 2006 ) . respiratory syncytial virus ( rsv ) is a single - stranded negative sense rna virus in the paramyxoviridae family that is the primary cause of morbidity and life - threatening lower respiratory tract disease in infants and young children worldwide , as well as an important pathogen of the elderly and immune compromised ( bader and mckinsey 2005 ; ebbert and limper 2005 ; mejias et al 2005 ) the disease burden associated with rsv infection is considerable as rsv is a leading cause of hospitalization for infants and young children worldwide having infection rates approaching 70%80% in the first year of life with many patients requiring hospitalization ( mccarthy and hall 2003 ; leung et al 2005 ) .
unfortunately , despite five decades of research , no safe and effective rsv vaccine is available and few effective prophylactic or therapeutic treatments are available .
thus , there is a critical need for the development of novel tools and platforms to augment virus diagnosis and foundational studies required for development of disease intervention strategies .
the surface topography of viruses lends to detection by antibodies , a feature that has been successfully used for bio - conjuagted np detection of rsv ( agrawal et al 2005 , 2006 ; bentzen et al 2005 ) .
rsv has two major surface proteins , ie , g and f proteins , that are primarily responsible for virus attachment and fusion , respectively ( tripp 2005 ) .
both g and f proteins are recognized by the immune system as neutralizing antigens ; thus a majority of antibodies are directed toward these proteins ( tripp 2005 ) .
the rsv f protein is more conserved than the g protein among rsv strains ( tripp 2005 ) , thus antibodies reactive to the f protein are ideal target developing bioconjugated nanoparticles . taking advantage of the composition - tunable emission of nps and their multivalent specificity
when conjugated to monoclonal antibodies , we tested the ability of these bioconjugated nps to detect rsv infection in vitro and in vivo using in a single - step format . we show that bioconjugated nps can be used to rapidly and sensitively detect rsv infection in both cell lines and mice , and that the detection can be done in a single step format .
this single - step format dramatically improves virus detection time , potentially saves costs , and reduces background staining that is currently associated with conventional virus detection methods .
to develop bioconjugated nanoparticles to detect rsv infection , semiconductor cadmium telluride ( cdte ) quantum dots ( qds ) were synthesized as previously described ( wang et al 2002 ; liu , chen , et al 2006 ) .
qds have been reported to be an excellent quantum particle for use in biological systems based on very high photoluminescence quantum efficiencies , and the ability to cover the whole visible spectral range depending on particle size ( liu , chen , et al 2006 ) .
thus , two different sized cdte qds with emission peaks at 585 nm and 540 nm were prepared ( figure 1 ) and used in the preparation of bioconjugated nanoparticles ( nps ) for detection of rsv infection .
these particles were prepared for bioconjugation as previously described ( wang et al 2002 ) , and conjugated to monoclonal antibodies reactive to rsv f protein , clone 131 - 2a .
briefly , thioglycolate ( tga)-coated cdte particles were mixed 1:1 with rsv anti - f protein monoclonal antibody ( 4 mg / ml ) and the solution was stabilized with 10x phosphate - buffered saline ( pbs ) , ph 7.2 to a final 1 x pbs concentration .
edc / nhs ( 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hcl / n - hydroxysulfosuccinimide ) was added to bring to qd solution to a final concentration of 50 mm/5 mm , respectively .
the reaction was incubated for 2 h at room temperature , then overnight at 4 c .
precipitated bioconjugated np aggregates were separated by centrifugation to remove unwanted aggregates from single , bioconjugated nanoparticles .
the final concentration of the purified np - antibody conjugate stock solution was 0.625 mm .
vero cells ( african green monkey kidney cells ) were grown in dulbecco s modified eagle s media ( dmem ) containing 10% fetal bovine sera ( dmem-10% ) .
for some experiments involving nps , vero cells were propagated on lab - tek chamber slides to 80%90% confluency using dmem-10% . respiratory syncytial virus strain a2 ( rsv )
rsv was diluted in dmem and the cells infected at a multiplicity of infection ( moi ) of 1 .
the virus was allowed to adsorb for 2 h at 37 c after which dmem-10% was added and the cells incubated at 37 c for 4 days . at day 4 post - infection ( pi ) ,
the virus was recovered by removing the cell culture supernatant , freeze - thawing the infected cells , and centrifuging the cell lysate to remove debris and recover the virus from the cell lysate supernatant .
the bioconjugated nanoparticles conjugated to anti - rsv f protein monoclonal antibody ( rsv - nps ) , ie , both 585 nm and 540 nm qds , were tested for their ability to detect rsv infection of vero cells .
rsv - infected vero cells were propagated on lab - tek chamber slides to 80%90% confluency and subsequently infected with rsv at a moi = 1 as previously described ( tripp et al 1999 ) . at day
4 pi , the media from the cells was removed , the cells were washed gently with pbs , fixed with acetone : methanol ( 60:40 ) , and air dried .
rsv - nps ( diluted 1:10,000 ) were added to the cells and incubated for 1 hour at 37 c , followed by three washes with pbs/0.05% tween 20 .
cells were visualized by fluorescent microscopy ( 40x ) on an olympus ckx41 inverted microscope ( olympus , center valley , pa ) , and photographed with an olympus q - color3 digital camera .
the most widely used assay to quantitate virus titers in both the laboratory and clinical setting is the virus plaque assay .
virus plaques on the cell lawn are enumerated by immunostaining to specifically detect virus plaques due to confounding background staining that is often associated with the counterstain procedure .
rsv - nps were evaluated for detection of rsv infection using a modification of an immunostaining plaque assay as previously described ( tripp et al 1999 ) .
briefly , the modification involved substituting rsv - nps in place of the primary ( anti - rsv monoclonal antibody : clone 131 - 2a ) , secondary ( anti - mouse whole molecule immunoglobulin g ( igg)-alkaline phosphatase conjugated ) , and detection substrate , eg , vector black alkaline phosphatase , to generate a single - step nanoparticle - based assay . in this modified assay , vero cells were plated onto 24-well flat - bottom plates 24-h prior to rsv infection .
subsequently , the cell supernatants were removed and rsv diluted in dmem was added to the cells at a moi = 1 .
the virus was allowed to adsorb for 1 h at 37 c after which the cells were overlayed with 2% methylcellulose in dmem-10% as previously described ( tripp et al 1999 ) , and cells incubated for 6 days at 37 c , 5% co2 . at day
6 pi , the overlays were removed , and cells were fixed with acetone : methanol ( 60:40 ) , and allowed to air dry .
cells were blocked for 30 minutes with powerblock ( 10% casein in pbs ) at room temperature .
rsv - nps , either 540 nm or 585 nm bioconjugated qds , were diluted 1:1,000 from 0.625 mm stock solution in pbs . for positive control wells ,
anti - rsv monoclonal antibody ( clone 131 - 2a ) was diluted 1:500 in pbs containing powerblock , added to the appropriate wells , and incubated 1 hour for rsv - nps , or 2 hours for the primary monoclonal antibody at 37 c .
cells were washed 3 times with pbs/0.05% tween . for standard immunostaining plaque assay , secondary antibody ( anti - mouse igg - alkaline phosphatase conjugated ) was diluted 1:500 in powerblock , added to cells , incubated for 1 hour at 37 c , washed twice with pbs , and developed using a vector black alkaline phosphatase substrate kit according to the manufacturer s protocol . for rsv - nps
, the plates were directly scanned for virus plaques using an amersham biosciences typhoon 9210 scanner with a fluorescence excitation ( 532 nm ) and emission ( 526 sp ) filter .
the virus plaques were enumerated and compared to plaque numbers obtained by standard counting using an olympus sz dissecting scope .
to determine the ability of rsv - nps to detect rsv infection in lung tissue , the natural site of rsv infection and replication ( tripp 2005 ) , 68 week old female balb / c mice were intranasally infected with 10 plaque - forming units ( pfu ) of rsv as previously described ( tripp et al 1999 ) . at day
4 pi , rsv - infected or nave balb / c mice were intravenously administered by tail vein 0.05 ml of rsv - nps , either 540 nm or 585 nm bioconjugated qds , diluted 1:1000 in pbs from a 0.625 mm stock solution . at day
5 pi , the lungs from rsv - np - treated or pbs carrier - treated mice were removed and prepared for thin sectioning and immunohistochemistry ( ihc ) analysis as previously described ( li , sarmento , et al 2005 ) . briefly
, lung tissue was frozen as tissue blocks and sectioned ( 20 m ) on a cryostat .
the frozen sections were placed on slides which were immersed in acetone at 4 c for 15 min , air dried for 30s , and then stored in pbs until immunostaining was performed .
briefly , slides were stained with anti - rsv monoclonal antibody ( clone 131 - 2a ) diluted 1:500 in pbs and incubated for 2 h at 37 c .
the slides were washed 2 times with pbs , and a secondary antibody ( fitc conjugated anti - mouse igg ) diluted 1:500 in pbs was added to slides and incubated for 1 h at 37 c , and washed twice with pbs .
the lung sections from rsv - np - treated mice and untreated mice that were immunostained as noted above were directly visualized using a fluorescent microscope .
to develop bioconjugated nanoparticles to detect rsv infection , semiconductor cadmium telluride ( cdte ) quantum dots ( qds ) were synthesized as previously described ( wang et al 2002 ; liu , chen , et al 2006 ) .
qds have been reported to be an excellent quantum particle for use in biological systems based on very high photoluminescence quantum efficiencies , and the ability to cover the whole visible spectral range depending on particle size ( liu , chen , et al 2006 ) .
thus , two different sized cdte qds with emission peaks at 585 nm and 540 nm were prepared ( figure 1 ) and used in the preparation of bioconjugated nanoparticles ( nps ) for detection of rsv infection .
these particles were prepared for bioconjugation as previously described ( wang et al 2002 ) , and conjugated to monoclonal antibodies reactive to rsv f protein , clone 131 - 2a .
briefly , thioglycolate ( tga)-coated cdte particles were mixed 1:1 with rsv anti - f protein monoclonal antibody ( 4 mg / ml ) and the solution was stabilized with 10x phosphate - buffered saline ( pbs ) , ph 7.2 to a final 1 x pbs concentration .
edc / nhs ( 1-ethyl-3-(3-dimethylaminopropyl ) carbodiimide hcl / n - hydroxysulfosuccinimide ) was added to bring to qd solution to a final concentration of 50 mm/5 mm , respectively .
the reaction was incubated for 2 h at room temperature , then overnight at 4 c .
precipitated bioconjugated np aggregates were separated by centrifugation to remove unwanted aggregates from single , bioconjugated nanoparticles .
the final concentration of the purified np - antibody conjugate stock solution was 0.625 mm .
vero cells ( african green monkey kidney cells ) were grown in dulbecco s modified eagle s media ( dmem ) containing 10% fetal bovine sera ( dmem-10% ) .
for some experiments involving nps , vero cells were propagated on lab - tek chamber slides to 80%90% confluency using dmem-10% . respiratory syncytial virus strain a2 ( rsv ) was propagated in vero cells as previously described ( tripp et al 1999 ) .
rsv was diluted in dmem and the cells infected at a multiplicity of infection ( moi ) of 1 .
the virus was allowed to adsorb for 2 h at 37 c after which dmem-10% was added and the cells incubated at 37 c for 4 days . at day 4 post - infection ( pi ) ,
the virus was recovered by removing the cell culture supernatant , freeze - thawing the infected cells , and centrifuging the cell lysate to remove debris and recover the virus from the cell lysate supernatant .
the bioconjugated nanoparticles conjugated to anti - rsv f protein monoclonal antibody ( rsv - nps ) , ie , both 585 nm and 540 nm qds , were tested for their ability to detect rsv infection of vero cells .
rsv - infected vero cells were propagated on lab - tek chamber slides to 80%90% confluency and subsequently infected with rsv at a moi = 1 as previously described ( tripp et al 1999 ) . at day
4 pi , the media from the cells was removed , the cells were washed gently with pbs , fixed with acetone : methanol ( 60:40 ) , and air dried .
rsv - nps ( diluted 1:10,000 ) were added to the cells and incubated for 1 hour at 37 c , followed by three washes with pbs/0.05% tween 20 .
cells were visualized by fluorescent microscopy ( 40x ) on an olympus ckx41 inverted microscope ( olympus , center valley , pa ) , and photographed with an olympus q - color3 digital camera .
the most widely used assay to quantitate virus titers in both the laboratory and clinical setting is the virus plaque assay .
virus plaques on the cell lawn are enumerated by immunostaining to specifically detect virus plaques due to confounding background staining that is often associated with the counterstain procedure .
rsv - nps were evaluated for detection of rsv infection using a modification of an immunostaining plaque assay as previously described ( tripp et al 1999 ) .
briefly , the modification involved substituting rsv - nps in place of the primary ( anti - rsv monoclonal antibody : clone 131 - 2a ) , secondary ( anti - mouse whole molecule immunoglobulin g ( igg)-alkaline phosphatase conjugated ) , and detection substrate , eg , vector black alkaline phosphatase , to generate a single - step nanoparticle - based assay . in this modified assay
, vero cells were plated onto 24-well flat - bottom plates 24-h prior to rsv infection .
subsequently , the cell supernatants were removed and rsv diluted in dmem was added to the cells at a moi = 1 .
the virus was allowed to adsorb for 1 h at 37 c after which the cells were overlayed with 2% methylcellulose in dmem-10% as previously described ( tripp et al 1999 ) , and cells incubated for 6 days at 37 c , 5% co2 . at day
6 pi , the overlays were removed , and cells were fixed with acetone : methanol ( 60:40 ) , and allowed to air dry .
cells were blocked for 30 minutes with powerblock ( 10% casein in pbs ) at room temperature .
rsv - nps , either 540 nm or 585 nm bioconjugated qds , were diluted 1:1,000 from 0.625 mm stock solution in pbs . for positive control wells ,
anti - rsv monoclonal antibody ( clone 131 - 2a ) was diluted 1:500 in pbs containing powerblock , added to the appropriate wells , and incubated 1 hour for rsv - nps , or 2 hours for the primary monoclonal antibody at 37 c .
secondary antibody ( anti - mouse igg - alkaline phosphatase conjugated ) was diluted 1:500 in powerblock , added to cells , incubated for 1 hour at 37 c , washed twice with pbs , and developed using a vector black alkaline phosphatase substrate kit according to the manufacturer s protocol . for rsv - nps
, the plates were directly scanned for virus plaques using an amersham biosciences typhoon 9210 scanner with a fluorescence excitation ( 532 nm ) and emission ( 526 sp ) filter .
the virus plaques were enumerated and compared to plaque numbers obtained by standard counting using an olympus sz dissecting scope .
to determine the ability of rsv - nps to detect rsv infection in lung tissue , the natural site of rsv infection and replication ( tripp 2005 ) , 68 week old female balb / c mice were intranasally infected with 10 plaque - forming units ( pfu ) of rsv as previously described ( tripp et al 1999 ) . at day
4 pi , rsv - infected or nave balb / c mice were intravenously administered by tail vein 0.05 ml of rsv - nps , either 540 nm or 585 nm bioconjugated qds , diluted 1:1000 in pbs from a 0.625 mm stock solution . at day
5 pi , the lungs from rsv - np - treated or pbs carrier - treated mice were removed and prepared for thin sectioning and immunohistochemistry ( ihc ) analysis as previously described ( li , sarmento , et al 2005 ) .
briefly , lung tissue was frozen as tissue blocks and sectioned ( 20 m ) on a cryostat .
the frozen sections were placed on slides which were immersed in acetone at 4 c for 15 min , air dried for 30s , and then stored in pbs until immunostaining was performed .
briefly , slides were stained with anti - rsv monoclonal antibody ( clone 131 - 2a ) diluted 1:500 in pbs and incubated for 2 h at 37 c .
the slides were washed 2 times with pbs , and a secondary antibody ( fitc conjugated anti - mouse igg ) diluted 1:500 in pbs was added to slides and incubated for 1 h at 37 c , and washed twice with pbs .
the lung sections from rsv - np - treated mice and untreated mice that were immunostained as noted above were directly visualized using a fluorescent microscope .
current antibody - based methods used for virus detection are cumbersome , have limited sensitivity due to substantial background staining , and are costly as they often require multiple antibodies and enzyme substrate for detection . in an effort to ameliorate these issues , and reduce the time for virus detection , we synthesized semiconductor cdte qds with emission at either 540 nm or 585 nm to be conjugated to monoclonal antibodies to detect rsv - infected cells .
these bioconjugated nps ( rsv - nps ) that are specific to rsv f protein were tested for their ability to detect rsv infection of cells both in vitro and in vivo . as expected ,
no differences in virus detection were observed using either 540 or 585 nm nps . as shown in the high resolution transmission electron microscopy ( hrtem ) images in figure 1 ,
the green emission cdte nps had an average size of approximately 3 nm ( a ) and the orange cdte nps of 5 nm ( b ) .
given the reported difficulties conjugating proteins to cdte nps ( tan et al 2004 ; gao et al 2005 ; mcveigh 2006 ) , the cdte nps were produced with a thioglycolate coating to allow direct anti - rsv f protein monoclonal antibody conjugation via an edc / sulfo - nhs conjugation reaction .
these bioconju - gated nps ( rsv - nps ) were stable for > 1 year at 4 c , and did not precipitate in the pbs diluent at room temperature or at 4 c . to determine the ability of the rsv - nps to detect rsv infection , in vitro studies
were performed using rsv - infected vero cells . in these studies , we compared rsv detection using conventional antibody staining methods , ie , primary and secondary antibody staining plus enzyme substrate , to that of a single - step detection method using rsv - nps . conventional antibody staining methods produced substantial background staining in mock - infected vero cells ( figure 2a )
, however did detect rsv plaque formation in the vero cell lawn ( figure 2b ) .
in contrast , rsv - nps used in a single - step method produced little background ( figure 2c ) , and readily detected rsv plaques ( figure 2d ) .
these results show that rsv - nps can be used to detect rsv in a single - step fashion , and that this method can be used to greatly reduce the amount of time and reagent needed for rsv detection compared to conventional immunostaining procedures ( tripp et al 1999 ) . extending the in vitro detection findings ( figure 2 ) , we determined if rsv - nps could be used to quantitate rsv titers in a single step fashion , thereby shortening the detection time needed to quantitate virus titers by plaque assay ( figure 3 ) . in these studies
, we compared a conventional immunostaining plaque assay which requires 5 or 6 days of in vitro culture post - rsv infection ( tripp et al 1999 ) to rapid single - step detection by rsv - nps .
for comparison of the different plaque assay methods , virus titers were assessed at days 2 , 3 , 5 , or 6 pi .
the results showed that at days 5 or 6 pi , conventional immunostaining ( figure 3a ) and the single - step rsv - np detection method ( figure 3b ) were similarly sensitive for detecting rsv plaques in the vero cell lawn .
however , rsv - nps could detect rsv plaques earlier ( days 2 or 3 pi ) compared with conventional immunostaining which was comparatively ineffective ( figure 4 ) , indicating that the single - step rsv - nps procedure was more sensitive at detecting limiting levels of rsv .
this result is not unexpected given the multivalent properties of rsv - nps . to evaluate the efficacy of rsv - nps to detect rsv infection in vivo , balb / c
were either intranasally infected with 10 pfu rsv or mock - treated , and at day 4 pi , rsv - infected or mock - treated mice were intravenously administered 0.05 ml of rsv - nps from the stock solution ( 0.625 mm ) by tail vein . at day 5
pi , the lungs from rsv - np - treated or mock - treated mice were removed , frozen , and prepared for thin sectioning and ihc analysis using conventional methods , i.e. primary and secondary antibody staining , or directly visualized by fluorescence microscopy in the case of rsv - np treatment . treatment with rsv - nps provided clear and rapid detection of rsv - infected lung tissue ( figure 5a ) with very limited background staining as assessed in lung tissue from mock - treated mice similarly administered rsv - nps ( figure 5b ) .
importantly , the magnitude and location of rsv - nps staining along the epithelial cells of the alveoli in the lungs was similar to that of conventional ihc staining ( figure 5c ) , and the background staining for rsv - nps was lower in nave mice compared to conventional ihc staining ( figure 5d ) .
the selective staining by rsv - nps of only rsv - infected lung epithelial cells suggests antibody - mediated specific targeting to sites of infection .
these results suggest that rsv - nps may be useful to determine the progression of rsv infection in the lungs , and as a result , aid our understanding of sites of infection , and help to define disease intervention strategies that employ bioconjugated np targeting strategies .
the results from this study show that rsv - nps can be used to detect rsv infection both in vitro and in vivo and suggest that beyond diagnostics , bioconjugated nps may be useful for multiplexed detection of viral and/or host cell antigens , as well as for targeting sites of virus infection with antiviral agents that may be linked to the nps .
indeed , bioconjugated nps have been used to identify the presence and progression of rsv infection in a human epithelial cell line , ie , hep-2 cells ( bentzen et al 2005 ) , as well as used in a variety of intracellular tracking and related studies ( alivisatos et al 2005 ; bruchez 2005 ; gao et al 2005 ; hotz 2005 ; shenoy et al 2006 ; mcveigh 2006 ) .
importantly , we show here that rsv - nps can be applied as tools to enhance virus detection by increasing sensitivity of virus detection while decreasing the detection time .
application of rsv - nps for virus detection in animal models can provide a path to assist our ability to dissect important parameters of rsv disease pathogenesis such as determining sites of virus infection and virus load in the lungs of infected animals .
 the integration of nanotechnology with biology has produced major advances in molecular diagnostics , therapeutics , and bioengineering . 
 recent advances have led to the development of functionalized nanoparticles ( nps ) that are covalently linked to biological molecules such as antibodies , peptides , proteins , and nucleic acids . 
 these functionalized nps allow for development of novel diagnostic tools and methods , particularly for pathogens , as rapid and sensitive diagnostics are essential for defining the emergence of infection , determining the period that preventive measures should be applied , for evaluating drug and vaccine efficacy , and for controlling epidemics . in this study , we show that functionalized nps conjugated to monoclonal antibodies can be used to rapidly and specifically detect respiratory syncytial virus in vitro and in vivo . 
 these results suggest that functionalized nps can provide direct , rapid , and sensitive detection of viruses and thereby bridge the gap between current cumbersome virus detection assays and the burgeoning need for more rapid and sensitive detection of viral agents . 
Introduction
Experimental procedures
Bioconjugated nanoparticles
Vero cell and virus propagation
Detection of RSV infection in vitro
Detection of RSV infection in vivo
Results and discussion
Conclusions