gold nanorods ( gnrs ) are anisotropic particles 
 whose surface plasmon 
 modes can be tuned as a function of aspect ratio , with optical resonances 
 ranging from visible to near - infrared wavelengths .
gnrs 
 have been investigated as contrast agents for optical biomedical imaging 
 modalities such as optical coherence tomography and photoacoustic 
 tomography ; they are also capable of producing linear and two - photon 
 excited luminescence , with detection limits at the single - particle 
 level . the large absorption cross sections 
 of gnrs
can also generate localized photothermal effects , with application toward the release of molecular 
 cargo and hyperthermic effects on diseased cells and tissues .
these attributes have sparked a global effort to develop gnrs into 
 theranostic agents for nanomedicine .
one hurdle in the scalable manufacturing of gnr - based materials 
 is the efficient exchange and removal of cetyltrimethylammonium bromide 
 ( ctab ) , a micellar surfactant used in the batch synthesis of gnrs .
ctab is cationic and moderately cytotoxic ( although 
 not insupportably so ) , most of which can be removed 
 by multiple washes and exchanges with chemisorptive surfactants ( e.g. , 
 pegylated thiols or dithiocarbamates ) , phospholipids , or other 
 surface - active agents .
however , ctab - coated gnr dispersions 
 are frequently destabilized during surfactant exchange , resulting 
 in partial aggregation and low recovery yields .
furthermore , ligand - modified 
 gnrs are often contaminated with residual ctab , which can induce nonspecific 
 protein adsorption and cell uptake under physiological conditions , or produce surface charge defects in materials 
 applications .
we have previously shown that ctab - depleted gnr dispersions 
 can be prepared when using sodium polystyrenesulfonate ( na - pss ) as 
 a mild detergent ; nevertheless , the stability 
 of such suspensions remains capricious in subsequent manipulations . 

we thus sought to develop a practical method for producing ctab - free 
 gnr dispersions that would be universally compatible with surface 
 conjugation protocols . in this letter
we describe an efficient 
 method for converting ctab - stabilized 
 gnrs into citrate - stabilized gnrs ( cit - gnrs ) via intermediate treatment 
 with pss .
the surface exchange process was monitored in stages by 
 x - ray photoelectron spectroscopy ( xps ) , an invaluable tool for quantitative 
 elemental analysis of surface adsorbates , as well as by attenuated 
 total reflectance infrared ( atr - ir ) spectroscopy and surface - enhanced 
 raman scattering ( sers ) .
our analyses indicate that ctab removal by 
 pss treatment is highly efficient , as is the subsequent displacement 
 of pss by citrate .
the cit - gnrs are fully dispersible in low - salt 
 solutions , and are easily functionalized by further ligand exchange 
 ( discussed in a companion article ) .
a six - stage 
 protocol was developed to convert ctab - gnrs ( 68  
 26 nm , lpr = 713 nm ) into cit - gnrs , using standard 
 ultrafiltration and centrifugation equipment .
ctab - gnrs were prepared 
 on a gram scale by the method described by khanal and zubarev , and diluted 4-fold to prevent premature flocculation 
 of ctab - gnrs during the initial purification stages . in a typical 
 process ,
a suspension of gnrs ( optical density ( o.d . ) = 3.2 ) stabilized 
 in 25 mm ctab was concentrated by stirred ultrafiltration to remove 
 excess surfactant , then diluted with deionized water ( stage 1 ; figure 1 ) .
the gnrs were then subjected to three cycles 
 of centrifugation and redispersion ( c / r ) to deplete ctab to trace 
 levels , using 0.15 wt % na - pss ( mw = 70 
 kda ; stages 24 ) .
we note that the residual ctab in the pss - stabilized 
 gnr suspension after stage 4 is < 1 m based on simple dilution 
 factors ; however , the free ctab concentration is likely even lower 
 due to its favorable adsorption to pss .
the pss - gnrs were then subjected to two additional c / r cycles using 
 5 mm sodium citrate ( stages 5 and 6 ) for exchange with pss , yielding 
 stable dispersions of cit - gnrs .
a complete procedure with exact volumes 
 and concentrations is provided in the supporting 
 information .
process flow diagram for converting ctab - gnrs into cit - gnrs 
 by 
 ultrafiltration ( stage 1 ) with successive c / r cycles using 0.15 wt 
 % na - pss ( stages 24 ) and 5 mm na3-cit ( stages 5 
 and 6 ) , with estimated surfactant concentrations after each stage .
the conversion 
 of ctab - gnrs into cit - gnrs was achieved with an 
 overall efficiency of 7585% , based on initial and final o.d . 

the main source of loss is due to incomplete 
 recovery of gnrs during the first two c / r cycles .
absorption spectroscopy 
 and transmission electron microscopy ( tem ) analysis of the citrate - stabilized 
 gnrs indicated essentially no changes in optical resonance ( max = 710 nm ; figure 2 ) or size distribution 
 ( figure s1 , supporting information ) .
on 
 the other hand , the exchange of pss to citrate was readily discernible 
 by electrokinetic measurements : the zeta potential of gnrs in dilute 
 na - pss solution ( 0.25 mg / ml , ph 5 ) was 55.6  8.3 mv , 
 while that of cit - gnrs in dilute phosphate buffered solution ( pbs ) 
 adjusted to ph 9.5 ( i  8.2 mm ) was less negative 
 ( mean 26.9  13.2 mv ; mode 21.1  7.1 mv ) . 

the cit - gnr suspensions are stable for months at low ionic strength 
 but aggregate if dispersed in highly saline solutions ( also see below ) , 
 and thus behave similarly as other citrate - stabilized au nanoparticles .
normalized 
 absorption spectra of gnr dispersions after ultrafiltration 
 ( stage 1 , red ) , c / r cycles with na - pss ( stage 3 , green ) , and c / r cycles 
 with na3-cit ( stages 5 and 6 , blue ) .
the incubation of gnrs with 70-kda na - pss ( minimum 1 h , prior 
 to 
 each c / r step ) is essential for producing well - dispersed cit - gnrs . 

previous studies in our laboratory have established that pss is useful 
 as a detergent for removing residual ctab from gnr dispersions , below 
 the level of observable cytotoxicity . however , na - pss by itself is not suitable as a peptizing agent : 
 it adsorbs weakly onto gnr surfaces in the absence of ctab , resulting 
 in metastable dispersions whose relative stability depends on polymer 
 concentration , suggestive of depletion effects .
gnrs cleansed with pss can be stabilized afterward by introducing 
 surface - active agents that do not associate strongly with the polyelectrolyte . 
 in this regard ,
polyanions with moderate binding activity such as 
 citrate are ideal for surface exchange ; the pss is gently displaced 
 from the gnrs while maintaining a negative zeta potential during the 
 exchange process .
nonionic , hydrophilic surfactants can also be used 
 to displace pss from gnr surfaces , but 
 the efficiency of exchange depends on the specific qualities of the 
 surface - active agent .
attempts to treat ctab - gnrs directly with citrate 
 without na - pss cleansing invariably led to rapid aggregation , regardless 
 of surfactant concentrations ( figure s2 , supporting 
 information ) .
this is not surprising , as the citrate anions 
 neutralize the positive surface charge supported by the resident ctab 
 layer , resulting in particle destabilization prior to complete ligand 
 exchange .
it has been reported that citrate preferentially adsorbs 
 onto the ends of ctab - gnrs , resulting in their end - to - end aggregation .
the ligand exchange process from stage 
 3 ( pss wash # 2 ) to stage 
 6 ( citrate wash # 2 ) was evaluated by xps , based on the binding energies 
 ( b.e . ) of various atomic species ( table 1 and 
 figure 3 ) .
the xps signal corresponding to 
 bromine ( br 3d ; 68.5 ev ) could not be detected even at stage 3 , whereas 
 that of nitrogen ( n 1s ; 401 ev ) was reduced below the limit of detection 
 by stage 5 , establishing the essentially complete displacement of 
 ctab from the gnr surfaces .
the xps signals for carbon ( c 1s ) , oxygen 
 ( o 1s ) , sodium ( na 1s ) , and sulfur ( s 2p ) were also greatly reduced 
 upon washing with 5 mm citrate ( stage 5 ) , indicating the gross displacement 
 of na - pss from the gnr surfaces ( figure 3a ) . 

the s 2p signal was almost completely gone by the second citrate wash 
 ( stage 6 ) , and below trace levels in the supernatant .
high - resolution 
 analysis of the c 1s region further revealed that citrate exchange 
 was accompanied by a change in the population of carbon subtypes , 
 with a reduction in hydrocarbon species ( c  h / c  c ; b.e . 

= 284.8 ev ) and an increase in the density of carboxyl groups ( c(=o)o ; 
 b.e .
= 288.2 ev ) , corresponding with the adsorption of citrate ( figure 3b ) .
binding energies from survey xps 
 spectra . at or below limit 
 of detection .
( a ) survey xps spectra 
 acquired from pelleted gnr samples after 
 stage 4 ( pss wash # 3 ) and stage 5 ( citrate wash # 1 ; 5 magnification ) , 
 indicating the disappearance of the n 1s signal .
( b ) high - resolution 
 ( regional ) xps spectra of the c 1s region , indicating relative changes 
 in carbon subtype after treatment with citrate .
it is worth noting that the xps spectra also indicated the 
 significant 
 presence of silver ( ag 3d ; ca .
25% versus au 4f ) , which remained approximately 
 constant throughout the ligand exchange process .
agno3 is 
 used as an additive in the seeded growth of gnrs , and has been postulated to contribute toward 
 shape control by its selective deposition onto longitudinal facets .
this notion has recently been brought into question 
 by high - resolution tem imaging using energy dispersive x - ray spectroscopy , 
 which indicated the distribution of ag over the entire gnr surface .
regardless of mechanism , the intensity of the 
 ag 3d xps signal in our study also suggests that the deposited ag 
 is mostly localized near the gnr surface .
the ligand exchange 
 process was also characterized by atr - ir spectroscopy 
 and sers , to identify the molecular species associated with the gnr 
 samples ( figures s3 and s4 , supporting information ) .
ir analysis of pelleted gnr samples from stages 1 , 3 , and 4 confirmed 
 the presence ( and depletion ) of ctab and pss , with the former producing 
 strong bands associated with sp c 
h 
 stretching ( 28002900 cm ) , ch3 deformation ( 14501480 cm ) , and 
 c 
n stretching ( 900950 cm ) , and 
 the latter producing peaks associated with the so3 group ( 10201200 cm ) .
pss displacement by citrate anions 
 produced additional spectral changes , dominated by co2 stretching ( 1575 cm ) and ch2 deformation ( 1390 cm ) .
sers analysis 
 also confirmed that pss was adsorbed onto gnrs by the enhanced so3 raman signal , but was displaced after 
 two washes with na3-cit .
overall , these vibrational analyses 
 further support the conclusions drawn from the xps data . citrate - stabilized 
 au nanoparticles are widely appreciated for 
 their versatility in surface modification protocols , with minimal 
 interference by the displaced electrolyte .
we conducted a preliminary survey of cit - gnrs dispersed in different 
 aqueous solutions , to determine their relative stability during surface 
 functionalization .
dispersions of cit - gnrs are stable in 10 mm sodium 
 citrate ( i = 60 mm ) , but slowly aggregate in standard 
 pbs ( i 160 mm ) or 1 m nacl . however , cit - gnrs 
 treated with nonionic surfactants such as tween-20 , chemisorptive 
 surfactants such as thiolated polyethylene glycol , or serum proteins 
 such as albumin all form stable dispersions in pbs . such coated gnrs 
 have been characterized and are further described in our companion 
 paper .
the protocol 
 described above is a mild and general approach for 
 depleting ctab and other cationic surfactants from aqueous nanoparticle 
 suspensions , allowing their clean exchange with citrate ions .
the 
 citrate - stabilized gnrs are compatible with surface conjugation chemistries 
 commonly applied to metal colloids , and provide a reliable platform 
 for making rational changes in chemical and biological properties 
 as a function of surface modification .
 stable 
 aqueous dispersions of citrate - stabilized gold nanorods 
 ( cit - gnrs ) have been prepared in scalable fashion by surfactant exchange 
 from cetyltrimethylammonium bromide ( ctab)-stabilized gnrs , using 
 polystyrenesulfonate ( pss ) as a detergent . 
 the surfactant exchange 
 process was monitored by infrared spectroscopy , surface - enhanced raman 
 scattering ( sers ) , and x - ray photoelectron spectroscopy ( xps ) . 
 the 
 latter established the quantitative 
 displacement of ctab ( by pss ) and of pss ( by citrate ) . 
 the cit - gnrs 
 are indefinitely stable at low ionic strength , and are conducive to 
 further ligand exchange without loss of dispersion stability . 
 the 
 reliability of the surface exchange process supports the systematic 
 analysis of ligand structure on the hydrodynamic size of gnrs , as 
 described in a companion paper . 
Introduction
Experimental Section
Results and Discussion
Outlook