quantum dots   ( qdots ) are nanometer - sized semiconductor 
 crystals that have   unique physical properties that differ from bulk material .  
 the fluorescent properties of qdots have been widely described , and numerous 
 applications based upon these fluorescent properties have been reported . in addition , previous studies have reported the 
 properties of many varieties of qdots [ 18 ] . of these , the most widely studied are cadmium 
 selenide / zinc sulfide core - shell nanocrystals . these consist of a semiconductor 
 core of cadmium selenide encapsulated in a multilayer shell of zinc sulfide 
 doped with cadmium . the shell passivates the surface of the core , 
 and the band gap is wider than that of the core , enabling quantum confinement 
 of an electron - hole pair generated in the core after photoexcitation . 
 ultimately , the electron hole pair recombines , resulting in a fluorescent 
 emission of a lower - energy photon in the visible region of the spectrum . the energy of the emitted photon is determined by the size of the quantum 
 confinement ( or the size of the qdot ) . qdots have several advantages over conventional 
 fluorescent dyes ; these include increased photostability , increased brightness , 
 quantum yields in excess of 8090% [ 1 , 9 , 11 ] , and a narrow emission 
 spectrum ( less than 30  nm full width at half - maximum in commercial products ) [ 1215 ] . furthermore , their multivalent surfaces 
 enable the attachment of more than one type of ligand or multiple copies of a 
 ligand to a single qdot . since their introduction into biology as imaging agents in 
 1998 [ 16 , 17 ] , qdots have increasingly found applications as fluorescent 
 probes in biology . to be useful as fluorescent probes in biological systems , 
 qdots must be soluble in water and commonly used buffers . additionally , they 
 must have colloidal stability and low nonspecific adsorption to cellular 
 membranes . these properties have been achieved using a number of techniques , 
 including encapsulation in micelles , silanization ,   encapsulation 
 in amphiphilic polymers [ 20 , 21 ] , and encapsulation in proteins such as 
 streptavidin . to further reduce nonspecific adsorption to cellular 
 membranes , a number of techniques may be used to modify the surface chemistry 
 of qdots . for example , we have recently demonstrated that nonspecific binding 
 can be significantly reduced by attaching polyethylene glycol chains ( i.e. , 
 by pegylating ) qdots coated in an amphiphilic modified polyacrylic acid polymer 
 ( amp ) . the length of the peg chain and the peg loading were demonstrated 
 to be important in reducing nonspecific adsorption to cellular membranes . when 
 pegs with short ( less than 12 ) repeat units were conjugated to qdots , a small 
 reduction in nonspecific adsorption to cellular membranes was observed . figure 1 shows the 
 effects of pegylation    ( peg2000 ) on nonspecific adsorption to 6 different cell 
 types . these cells were treated with a 50  nm solution of pegylated amp - coated 
 qdots or a 50  nm solution of amp - coated qdots . a significant reduction in 
 nonspecific adsorption to cellular membranes was obtained by the addition of 
 peg2000 . the nonspecific adsorption is cell - type specific , as can be seen in 
 the relatively low nonspecific adsorption of amp - coated qdots to the surfaces 
 of 3t3 cells compared to the high levels of nonspecific adsorption to hek 
 cells . in 
 addition to surface modification techniques such as pegylation , a wide variety 
 of biologically active molecules have been attached to qdots , including 
 proteins   [ 2431 ] ,   peptides [ 3234 ] , dna   [ 3543 ] , rna , peptide nucleic acid ( pna ) , cytokines [ 46 ] , viruses [ 47 ] , and antibodies 
 [ 4854 ] . the qdots - based imaging applications 
 that have been reported in the literature are extensive and encompass a wide 
 variety of imaging applications . of these , live cell imaging   and 
 whole animal imaging   have received a great deal of interest . in 
 addition to qdots that emit in the visible region of the electromagnetic 
 spectrum , near - infrared qdots have been developed that have a cadmium telluride 
 core instead of a cadmium selenide core . these near - ir dots have found 
 applications in the clinic as tools for imaging sentinel lymph nodes during 
 surgery . we are interested in using qdots that have been conjugated with small 
 molecules [ 5560 ] , antibodies ,   and peptides   to image receptors and transporters in cell cultures , oocytes , and , ultimately , 
 neurons . in our early work , we used qdots to image the serotonin transporter 
 ( sert ) using pegylated serotonin ligands   attached to the surfaces 
 of qdots via an acid - base interaction ( see figure 2 ) . these conjugates 
 antagonized the serotonin transporter protein ( sert ) with an ic50 of 
 115 m in transfected hek-293 cells . using these conjugates qdot 
 preparations that contain an amphiphilic coating on the qdots surface are commercially available , and 
 a variety of methodologies , including those involving sulfo - smcc   and adaptor proteins , have been used to conjugate ligands to the 
 coated qdot . our current strategy uses 
 commercially available qdots that have either an amphiphilic coating ( amp ) on 
 the surface of the dots , or amp qdots with an additional coating of 
 streptavidin . pegylated ligands may be attached to the surface of these dots 
 using two different methodologies . either they may be covalently attached to 
 the amp coating using 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide 
 hydrochloride ( edc ) coupling chemistry , or a biotinylated derivative of the 
 biologically active ligand may be attached to the surface of 
 streptavidin - coated qdots via a streptavidin - biotin interaction . using the 
 pegylated ligand approach , we have synthesized a novel qdot conjugate and tested 
 its binding activity to the gabac receptor , a ligand - gated ion 
 channel that is found in retina and other central nervous system tissue and 
 that is activated in vivo by -aminobutyric acid ( gaba ) . specifically , we have investigated a peg 
 derivative of muscimol , a known agonist of both gabac and gabaa receptors ( see figure 3 ) . multiple copies of this ligand have been conjugated 
 to the surface of amp - coated qdots and used to image gabac receptors 
 expressed in xenopus laevis oocytes . streptavidin - coated 
 qdots and amp - coated qdots with maximum emissions of 605 and 585  nm were 
 obtained from invitrogen ( carlsbad , calif , usa ) . n - hydroxy urea , dimethyl 
 acetylenedicarboxylate , 1,5-diazabicyclo[5.4.0 ] undec-7-ene ( dbu ) , borane 
 dimethyl sulfide , and n - hydroxy succinimide ( nhs ) were obtained from sigma - aldrich ( st.louis , mo , usa ) . trifluoroacetic acid ( tfa ) , potassium hydroxide , and 
 hydrazine monohydrate were obtained from vwr ( west chester , pa , usa ) . ( warrington , pa , usa ) , and sephadex g-50 was obtained from amersham biosciences 
 ( uppsala , sweden ) . t - butyloxacarbamate ( boc)-protected n - hydroxy 
 succinimide - activated peg3400 ester ( boc - peg - nhs ) was obtained from nektar 
 therapeutics ( huntsville , ala , usa ) . this 
 was then coupled to the peg linker via an aminohexanoyl nhs ester to give the 
 pegylated muscimol ligand . the ligand was characterized by matrix - assisted 
 laser desorption / ionization time - of - flight ( maldi - tof ) mass spectroscopy and 
 conjugated to amp - coated qdots via an edc coupling . dimethyl 
 acetylenedicarboxylate ( 3.1  ml ) was added dropwise to a solution of n - hydroxy 
 urea ( 1.9 g , 25 mmols ) and dbu ( 4.19  g , 28  mmols ) in methanol ( 25 ml ) at 0c . the 
 resulting solution was stirred at 0c for 10 minutes , and then evaporated under 
 reduced pressure . concentrated hydrochloric acid was added until a ph of 1 was obtained . this solution was extracted with diethyl 
 ether , dried over magnesium sulfate , filtered , and then evaporated . the 
 resulting solid was recrystallized from methylene chloride to yield 1.1  g of 
 methyl 3-hydroxy isoxazole-5-carboxylate ( i ) in a 32% yield . this was converted 
 to ( ii ) by stirring 0.84  g of ( i ) in ammonium hydroxide ( 3  ml ) and methanol ( 3  ml ) 
 for 1 hour , followed by recrystallization from ethanol to give 0.75  g of 
 3-hydroxyisoxazole-5-carboxamide ( ii ) in 88% yield as the ammonium salt . muscimol 
 ( iii ) was obtained from 1  g of ( ii ) by reduction with borane dimethyl sulfide in 
 tetrahydrofuran ( thf ) to give 0.2  g of ( iii ) in a 22% yield after purification 
 by ion exchange chromatography . the 
 synthetic route used to synthesize the pegylated muscimol ligand initially , the aminohexanoyl spacer was synthesized by reacting 6-amino 
 hexanoic acid with boc anhydride in methanol to give 6-(tert - butoxycarbonylamino)hexanoic acid this was converted to 2,5-dioxopyrrolidin-1-yl 6-(tert - butoxycarbonylamino)hexanoate 
 ( v ) by reacting ( iv ) with nhs in the presence of dicyclo carbodiimide ( dcc ) . the product was recrystallized from ether / hexanes resulting in a 38% yield of 
 ( v ) . this was coupled to muscimol in pyridine to give tert- butyl 
 6-((3-hydroxyisoxazol-5-yl)methylamino)-6-oxahexylcarbamate ( vi ) in a 54% 
 yield . the boc protecting group was removed using tfa to give 6-amino - n-((3-hydroxyisoxazol-5-yl)methyl)hexanamide 
 ( vii ) in a 100% yield . this was coupled to boc protected peg3400 nhs ester to give ( viii ) in 100% yield . the boc protecting group was removed using tfa to give ( ix ) . compounds ( viii ) and ( ix ) were 
 characterized by maldi - tof mass spectroscopy ( applied biosystems voyager mass 
 spectrometer equipped with a 337  nm nitrogen laser ) using an acceleration 
 voltage of 25  kv , and the spectra were obtained by averaging of 3064 scans . the samples were prepared using a saturated matrix stock solution , consisting 
 of 2,5-dihydroxybenzoic acid and 0.01  m sodium iodide dissolved in 
 methanol . the peg derivatives ( viii ) and 
 ( ix ) were prepared ( 5  mm ) in methanol . the sample was added to the matrix by 
 mixing the sample and stock solutions in a 2 : 5 : 2 ratio of sample to matrix to 
 salt ( v / v ) . mass calibration of the 
 instrument employed a peg standard , and was prepared using the same protocol as 
 that employed for the other samples . analysis of the resulting spectra 
 indicated that compounds ( viii ) and ( ix ) were polydisperse . compound ( viii ) exhibited masses ranging from 
 3241  da to 4188  da ( indicative of muscimol conjugation to pegs of different 
 lengths ) , and a primary peak at 3726  da . the treatment of ( viii ) with tfa to 
 yield compound ( ix ) resulted in a maldi - tof spectrum shift of 100  da ( primary 
 peak at 3626  da ) , consistent with loss of the boc protecting group . the ligand was conjugated to qdots using an edc coupling 
 in which 1000 equivalents of ligand were mixed with 750 equivalents of nhs and 
 edc in borate buffer at ph 8.5 . to this was added a solution of amp - coated 
 qdots ( 8.4  m ) . unbound ligand was 
 removed by sephadex g-50 chromatography . the coupling of amino - terminated 
 peg2000 to amp - coated qdots using edc has been studied in an earlier 
 publication , and the efficiency of coupling has been reported to be 20% when 
 2000 equivalents of methoxy - terminated aminopeg2000 are reacted with 1 equivalent 
 of amp - coated qdots . since the terminating muscimol of the present 
 ligand is attached to peg3400 , the coupling efficiency is likely to be similar . 
 on this basis , we estimate the number of muscimol ligands to be around 150200 
 per qdot . the derivatized qdots were characterized by 
 electrophoresis in 1% agarose gel ( see figure 4 ) . the gel demonstrates that the 
 muscimol - conjugated qdots ( lane 3 ) , as well as qdots conjugated with peg2000 ( lane 4 ) , have a wide 
 distribution in the number of ligands attached to their surface , as they streak 
 on the gel more than unconjugated qdots ( lane 2 ) . it is important to note that 
 mobility in the gel does not depend merely on mass , but rather on 
 mass - to - charge ratio . thus , despite the substantial difference in mass of the 
 peg2000 versus the muscimol - terminated peg3400 ligand , the bands representing 
 the qdots conjugates that contain ( numerous copies of ) these ligands exhibit 
 similar mobilities ( lanes 3 and 4 ) . the present experimental conditions ( 1% 
 agarose gel ) do not separate protein standards that span a molecular weight 
 range of 10250  kda ( data not shown ) . the oocytes   used in this study were obtained from adult 
 female x. laevis toads . the oocytes 
 were stored in physiological saline ( ringer solution ; 100  mm nacl , 2  mm kcl , 2  mm 
 cacl2 , 1  mm mgcl2 , 10  mm glucose , and 5  mm hepes , ph 7.4 ) . using previously reported procedures , we expressed gabac receptors ( human 1 and perch 1b ) in x. laevis oocytes [ 67 , 68 ] . crna 
 ( 50  nl ) for each of the receptor subunits was injected into the oocyte , and the 
 oocytes were assayed after 1872-hour 
 incubation in ringer solution containing 0.1  mg / ml gentamycin at 1619c to allow for expression 
 of the gabac receptors . oocyte imaging was carried out in a glass - bottom dish into which gabac expressing oocytes and oocytes 
 that did not express gabac were placed . these oocytes were incubated 
 for 510 minutes in a drop ( 25 l ) of solution containing either 34  nm 
 amp - coated qdots conjugated to the muscimol ligand , or 34  nm amp - coated qdots 
 that lacked conjugated muscimol ligand . the oocytes were then imaged using a 
 confocal microscope ( leica model dm - ire2 with 20x objective ) with excitation at 
 476  nm , and with detection of fluorescence emission over a wavelength range ( 580620  nm ) 
 that included the qdot emission peak 
 ( 605  nm ) . at the beginning of experiments conducted on a given day , we 
 established microscope settings relevant to excitation illumination and 
 detection of fluorescence emission ( gain and offset ) with use of either a human 
 1 gabac - expressing or perch 1b gabac - expressing oocyte incubated with 34 nm muscimol - conjugated amp - coated qdots . figure 5 shows the binding of muscimol - conjugated 
 amp - coated qdots and unconjugated amp - coated qdots to oocytes expressing the 
 human 1   gabac receptor , and to nonexpressing control oocytes ( see figure 5 legend ) . when gabac - expressing oocytes were incubated with a 34  nm solution of muscimol - conjugated dots for 10 minutes , a fluorescent halo was observed at the oocyte surface membrane ( panel a ) . the fluorescent image can be compared with the corresponding bright - field image 
 ( panel b ) , which shows the position and focus of the oocyte . by comparison with 
 panel a , no fluorescence halo was observed upon similar incubation of a 1   gabac - expressing oocyte with amp - coated qdots , that is , with a structure that lacked muscimol ( panel 
 c ) . halo fluorescence of the oocyte 
 surface membrane was also absent when a nonexpressing oocyte was incubated with 
 34  nm muscimol - conjugated dots ( panel e ) . these results indicate that the 
 muscimol ligand is necessary for binding of the conjugate to the oocyte surface 
 membrane . as noted in section 2.1.4 , the muscimol - conjugated 
 amp - coated qdot preparation used in the 
 oocyte imaging experiments contained 150200 muscimol - terminated chains per 
 qdot . in some preparations ( not illustrated ) of these muscimol - conjugated 
 amp - coated qdots , the extracellular medium surrounding the oocytes exhibited 
 aggregation of the fluorescent particles . in developing the method of 
 preparation of the conjugate , we observed that if 2000 equivalents of the 
 muscimol ligand were reacted with amp - coated qdots in the presence of 1500 
 equivalents of edc and nhs , aggregates formed that subsequently precipitated 
 from solution . it is 
 likely that this aggregation is due to hydrogen bonding between muscimol 
 ligands on adjacent qdots . the size and solubility of these aggregates likely 
 depended on the number of ligands conjugated to the qdots . to quantify the extent of binding of muscimol - conjugated 
 amp - coated qdots to the oocytes , we analyzed the surface membrane and 
 extracellular regions of a given fluorescence image . using metamorph software ( offline version 
 6.3r0 ; universal imaging corp . , downington , pa , usa ) , we determined the intensities of 
 pixels underlying a multisegmented line that traced the arc - like border of the 
 oocyte ( 1525 straight - line segments ; 450750 pixels ) , and tabulated the 
 resulting pixel values in relation to a 0255 gray scale . we similarly 
 determined the intensities of pixels that corresponded with an identical 
 multisegment line constructed within the extracellular region of the image ; 
 tabulated intensities for this control extracellular region were taken as a 
 measure of background ( i.e. , surround ) fluorescence . for the image shown in figure 5a , fluorescence intensities determined for the halo ( henceforth termed 
  border  ) at the oocyte surface membrane and the surrounding extracellular 
 medium ( background ) were 67.31  36.79 ( mean  sd ) and 22.30  21.18 , respectively . , results obtained in experiments similar in design to that 
 described in figures 5a , 5b ( human 1   gabac - expressing oocytes ; incubation with 34  nm muscimol - conjugated dots ) indicated a border fluorescence of 88.84  64.84 and a background fluorescence of 31.60  35.50 ( n = 11 ) , respectively . additional experiments of the same design ( not illustrated ) , conducted on oocytes expressing the perch 1b 
 receptor , yielded border and background fluorescence intensities of 
 109.58  58.42 and 18.54  16.47 aggregate results obtained in 4 experiments 
 in which gabac - expressing oocytes were incubated with unconjugated 
 amp - coated qdots ( see figures 5c , 5d ) yielded border and background fluorescence intensities of 15.79  23.18 and 13.13  18.17 , respectively . among 14 experiments that 
 involved the incubation of 34 nm muscimol - conjugated dots with nonexpressing 
 oocytes ( see figures 5e , 5f ) , border and background fluorescence 
 intensities were 15.14  22.35 and 16.78  22.17 , respectively , . two - way anova analysis of results 
 obtained with the muscimol - conjugated amp - coated qdots showed that for both human 1 gabac - expressing and perch 1b gabac - expressing oocytes , the 
 fluorescence intensity of the border differed significantly from that of the 
 background . for nonexpressing oocytes incubated with the conjugate in addition , 
 the treatment of gabac - expressing oocytes with free ( i.e. , 
 non - qdot - conjugated ) gaba , muscimol , or pegylated muscimol significantly 
 reduced binding of the muscimol - qdots conjugate to the oocyte surface membrane 
 ( see gussin et al .   for further details ) . the primary finding of the experiments involving the 
 incubation of muscimol - conjugated amp - coated qdots with gabac - expressing 
 oocytes is that these conjugates exhibit specific binding at gabac receptors . this binding depends on the presence of muscimol in the conjugate , as 
 ( unconjugated ) amp - coated qdots show no significant binding to oocytes 
 expressing gabac receptors . the approach described here builds on 
 our earlier work with pegylated serotonin attached to qdots in which we found 
 that these conjugates exhibited binding at serotonin transporters expressed in 
 hela and hek cells . these findings indicate that it is possible to specifically 
 label transporter proteins and ligand - gated receptors with qdots that have multiple 
 copies of a membrane receptor or membrane transporter ligand attached through a 
 peg linker . the presence of numerous copies of ligand in the muscimol - qdot conjugate described here raises the possibility that these conjugates 
 bind to multiple gabac receptors in a cross - linking fashion . indeed , 
 it is reasonable to hypothesize that the multiplicity of the muscimol ligand , 
 as well as the length of the peg linker that tethers each ligand to the qdot , 
 favors such cross - linking . however , experiments conducted to date , while 
 clearly establishing the ability of this conjugate to bind to 
 cell - surface - expressed gabac receptors , do not address the extent to 
 which receptor cross - linking affects this binding activity . other investigators 
 have used fluorescent probes to track the diffusion dynamics of single 
 receptors ( single - particle tracking ( spt ) ) , 
 and such an spt approach could be useful for evaluating the cross - linking 
 activity of the present muscimol - qdot conjugate . for example , dahan et al . have examined the 
 diffusion dynamics of glycine receptors in neuronal membranes by labeling the 
 receptor with a conjugate consisting of a primary antireceptor antibody , 
 biotinylated secondary antibody , and streptavidin - coated qdots . to test the 
 extent of receptor cross - linking by this conjugate , they investigated , as a 
 comparison system , an fab fragment of the primary antibody that had been linked 
 to an organic fluorophore ( cy-3 ) . dahan et 
 al .   found that the receptor dynamics determined with the qdot conjugate and the cy-3-containing 
 molecule were similar , indicating that neither the presence of the sa - qdots nor 
 some other feature of the qdot - containing conjugate promoted significant 
 receptor cross - linking . it should be emphasized that the structure of the 
 presently described muscimol - qdot conjugate ( see figure 2 ) differs from the 
 qdot - containing conjugate studied by dahan et al .   in several respects , including the presence 
 of a small - molecule ligand ( muscimol ) rather than an antibody as the 
 receptor - reactive moiety , a high valency ( copy number ) of ligands per qdot , and 
 a separation of each ligand from the qdot by a long linking chain ( peg3400 ) . in 
 future experiments , it may be possible to test for cross - linking by the 
 muscimol - qdot conjugate using an approach in which gabac receptor 
 dynamics determined with the muscimol - qdots conjugate are compared with those 
 determined using a structure optimized for spt , for example , a fluorescent 
 probe attached to a single receptor [ 26 , 27 ] .
we report a methodology for labeling the gabac receptor on the surface membrane of intact cells . 
 this work builds upon our earlier work with serotonin - conjugated quantum dots and our studies with pegylated quantum dots to reduce nonspecific binding . in the current approach , a pegylated derivative of muscimol 
 was synthesized and attached via an amide linkage to quantum dots coated in an amphiphilic polymer derivative of a modified polyacrylamide . 
 these conjugates were used to image gabac receptors heterologously expressed in xenopus laevis   oocytes .