the synthesis of high - quality 
 colloidal core  shell quantum 
 dots ( qds ) is usually performed in organic solvents . from these solvents , they can be processed and used in numerous 
 applications in optoelectronics and photovoltaics . alternatively , 
 the qds can be subsequently transferred into aqueous solutions in 
 order to increase their range of applications , particularly in biological 
 labeling and environmental sensing .
such a transfer requires either hydrophilic bifunctional ligands 
 to be exchanged for the native hydrophobic monofunctional coordinating 
 ligands , usually octadecylamine ( oda ) or the addition 
 of amphiphilic ligands or diblock copolymers where the hydrophobic 
 regions intercalate with the native ligands and hydrophilic regions 
 are exposed to the aqueous environment .
there are 
 advantages and disadvantages to each approach , but it mainly 
 comes down to a choice of size vs stability .
the long - chain native 
 ligands and the amphiphilic nature of ligands used in the intercalation 
 approach lead to qds with relatively large overall diameters .
the 
 ligand - exchange approach can , in principle , use extremely small ligands . 

however , when using these qds for fluorescence applications , it has 
 been found that fluorescence quenching depends on both ligand size 
 and the functional group used , so a balance 
 is usually found between size , stability , and fluorescence quantum 
 yield . as a compromise , small polydentate ligands such as dihydrolipoic 
 acid ( dhla ) have been used to improve colloidal stability over monodentate 
 ligands , although we have recently found 
 that the lower density of dhla ligands on the surface compared to 
 monodentate mpa ligands leads to higher nonspecific adsorption of 
 target thiol molecules at low concentrations . in any case , the colloidal stability of ligand - exchanged quantum 
 dots is usually lower than the amphiphilic polymer - coated qds and 
 depends strongly on the nature of the ligands and the solvent conditions . 

this can be a particularly significant problem at low qd concentration 
 and under reductive or oxidative conditions , as is often the case in biological fluorescence labeling applications . combining the advantages of ligand exchange using compact , water - soluble 
 ligands with cross - linking
has the potential to allow a better compromise 
 in which the size increase is modest while the improvement in colloidal 
 stability should be significant .
only a few examples of cross - linking 
 compact , ligand - exchanged qds have been reported , and they have used 
 a chemical cross - linking approach . however , a 
 major disadvantage with chemical cross - linking is that it is difficult 
 to inhibit interparticle cross - linking , which means that a purification 
 step may be required and overall yield will be compromised .
photochemical 
 cross - linking is an attractive alternative to localize the cross - linking 
 to within a particle , particularly if the cross - linking group is not 
 exposed to the solvent environment .
self - assembled monolayers ( sams ) 
 of diacetylene ligands on gold were directly photo - cross - linked using 
 high - energy ultraviolet light ( uv , 254 nm ) and were shown to result 
 in surfaces that are extremely robust for use in harsh environments . in this study , we demonstrate that qds themselves can initiate cross - linking 
 of bifunctional diacetylene ligands using lower energy visible light 
 to generate radicals ( scheme 1 ) . photoexcited 
 cdse has been previously used to generate radicals on adsorbed species 
 for charge separation in photovoltaic applications and in solid - state polymer  nanocomposite blends . however , to the best of our knowledge , using a qd - mediated photo - cross - linking 
 approach in solution to limit interparticle cross - linking while , at 
 the same time , promoting intraparticle cross - linking to improve the 
 colloidal stability of those same qds has not yet been demonstrated . 

we show that taking advantage of the qd as a visible - light initiator 
 leads to much better cross - linking of the ligands than direct cross - linking 
 of the diacetylene groups with uv light , since competing ligand dissociation 
 reactions induced by uv exposure are avoided .
our approach results 
 in extensive intraparticle cross - linking but with practically no interparticle 
 cross - linking , and more importantly , it leads to excellent long - term 
 colloidal stability of the qds in aqueous environments while maintaining 
 relatively small hydrodynamic sizes .
the synthesis of the 
 bifunctional diacetylene ( da ) ligand followed a combination of procedures 
 described by xu et al . and kim et al .
details of the da ligand synthesis and nmr peak 
 assignments are provided in the supporting information .
cdse / zns ( core / shell ) quantum dots ( em = 597 nm ) coated with hydrophobic octadecylamine ( oda ) ligands 
 were purchased from ocean nanotech ( springdale , ar ) as a dried powder 
 and dissolved in toluene ( emd chemicals inc . , billerica , ma ) .
prior 
 to the ligand exchange procedure , the as - purchased qds were purified 
 from excess ligands by precipitating from toluene with acetone ( emd ) , 
 centrifuging at 14  100 g ( 14  500 rpm , 
 minispin plus , eppendorf ) , and discarding the supernatant .
then , the 
 precipitated qds were redissolved into chloroform ( drisolv , emd ) , 
 mixed with methanol ( emd ) , and centrifuged again and the supernatant 
 was discarded .
a ligand solution was prepared by adding 10 mg of the 
 da ligand to 1 ml of chloroform or by adding 2.66 l of mercaptopropanoic 
 acid ( mpa ) ( alfa aesar , ward hill , ma ) in 1 ml of methanol .
the ph 
 was adjusted to 11 by adding microliter quantities of a stock solution 
 of tetramethylammonium hydroxide ( tmaoh ) ( alfa aesar ) in methanol . 

the ligand solution was added to the precipitated qds , and stirred 
 for 24 h at room temperature in the dark .
it has been shown that 254 nm 
 uv exposure can be used to photopolymerize 
 diacetylenes .
the fluorescence of the 
 sample as a function of 254 nm ( short wave ) uv exposure time was monitored 
 to determine the optimal cross - linking time in terms of the fluorescence 
 properties .
emission spectra were obtained by exciting at 530 nm in 
 a 100 l quartz fluorescence cuvette ( starna cells ) . from all 
 the samples , those exposed for 0 , 20 , 100 , and 210 min
were then analyzed 
 with an ft - ir spectrometer ( bruker , vertex 70 ) equipped with a dtgs 
 detector .
a 40 l aliquot of the sample was taken from the stock 
 solution at the specified time of exposure and placed in the exact 
 center of a caf2 window which was then left to dry under 
 a n2 atmosphere so that the ir beam diameter completely 
 probed the whole sample spot .
to transfer 
 the water - soluble qd - da and qd - mpa to water , methanol and acetone 
 were added , respectively , and the mixture was centrifuged to precipitate 
 the qds from the reaction solution .
having removed the supernatant , 
 the qds were redissolved into millipore ( 18.2 mcm ) water . 

each solution was transferred to a four - windowed quartz fluorescence 
 cuvette with a stopper ( starna ) for further analysis .
photographs to show 
 qd fluorescence and colloidal dispersion in 
 water were taken with a 10.1 mp camera ( pentax ) whereby a hand - held 
 uv lamp operating at 366 nm ( long wave ) was shone from beneath the 
 four - windowed quartz fluorescence cuvettes containing the samples . 

single particle burst analysis and fluorescence 
 lifetimes were acquired on a picoquant microtime 200 fluorescence 
 microscope as previously described .
a pulsed laser 
 operating at 485 nm , 15 w , and 5 mhz was used for excitation 
 and focused though the objective ( planapo 63xw , olympus ) to a diffraction - limited 
 spot .
the emission was collected by the same objective and passed 
 through a 100 m pinhole and a 585/55 m filter before being detected 
 on a single photon counting avalanche diode ( pdm series , microphotonic 
 devices , bolzano , italy ) , and the data was saved in time - tagged time - resolved 
 format to enable offline calculation of fluorescence bursts , fluorescence 
 lifetime , and fluorescence correlation spectroscopy ( fcs ) using the 
 symphotime software ( version 5.3.2 , picoquant gmbh ) .
the absorption and photoluminescence ( pl ) 
 spectra of the qds before 
 ligand exchange ( qd - oda ) and after ( qd - mpa , or qd - da ) are shown in 
 figure 1 .
in addition to the characteristic 
 qd exciton absorbance peak at 583 nm and the broad absorbance that 
 increases with increasing energy that is observed in each sample , 
 the absorbance of qd - da shows another intense peak between 400 and 
 430 nm , which is assigned to  * transitions in 
 the diacetylene moiety .
it was found that using the same initial qd 
 concentration for the ligand exchange resulted in better solubilization 
 efficiency compared to qd - mpa , as evidenced by the stronger color 
 of the solutions shown in the inset of figure 1 .
this could be a consequence of the diacetylene ligands favoring 
 a close - packing arrangement on the qd surface , resulting in a sam - driven 
 ligand exchange process , whereby hydrophobic interactions between 
 chains result in a higher ligand density compared to the shorter and 
 more soluble ( i.e. , labile ) mpa ligands . in both cases , however , the 
 fluorescence quantum yield decreased by approximately half , which 
 is common for thiolated ligands on qds  even those with a core 
the da ligands decreased 
 the quantum yield a little more than mpa , consistent with a more complete 
 ligand exchange ( more thiols ) because of sam packing . absorption and photoluminescence 
 spectra of qds before ( black ) 
 and after ligand exchange with mpa ( blue ) and da ( red ) .
inset : photographs 
 of qds under room light after ligand exchange using mpa ( left ) and 
 da ( right ) show relative solubilization efficiency .
we hypothesized that using high - energy uv light 
 to directly photo - cross - link 
 diacetylene ligands on qds would not be ideal , since the qd 
thiol 
 coordination bond would be prone to photooxidation but that qds excited with visible light could be used to initiate 
 photo - cross - linking . to test our hypothesis , we measured the absorption 
 spectra of qd - da in water as a function of time left exposed to room 
 light .
one qd - da sample was first exposed to 30 min of uv light , while 
 the other was not .
this data is presented in parts a and b of figure 2 , respectively . at day 0 ,
the characteristic diacetylene 
 peak between 400 and 430 nm was stronger for the unexposed qd - da compared 
 to the uv - exposed qd - da .
this could be a consequence of da ligands 
 undergoing partial cross - linking and/or uv - induced dissociation ( vide infra ) .
as the qds were left exposed to room light 
 for up to 2 weeks , significant changes in the absorption spectra were 
 observed for both samples , although the details are somewhat different . 

both samples show the formation of a new peak at 650 nm , which 
 begins to form within the first day and increases steadily .
this increase 
 is concomitant with a decrease in the absorbance in the 350450 
 nm region .
we assign this 650 nm peak to  * transition 
 of the long , conjugated -electron system that results from 
 extensive cross - linking of the da ligands on the qd induced by visible 
 light .
a similar peak has also been observed for similar ligands on 
 gold particles that were irradiated with uv light for 30120 
 min . in the case of the qds
studied here , 
 qd - da that had been exposed to uv light first showed an increase in 
 the 650 nm peak within 7 days and then saturated , indicating that 
 cross - linking stopped prematurely . for the qd - da samples
that had 
 not been exposed to uv light first , the 650 nm peak continued to increase 
 for the full 14 days and the decrease in the 350450 nm region 
 was much more pronounced , suggesting that visible - light - induced cross - linking 
 was slower but more extensive , without the competing process of uv - induced 
 ligand dissociation that prematurely halts cross - linking . in both 
 cases , the excitonic qd peak at 583 nm showed a moderate blue - shift : 
 to 572 nm in the case of uv - exposed qd - da and to 568 nm for the non - uv - exposed 
 qd - da .
it is interesting to note that , for the uv - exposed samples 
 on day 0 , the peak at 650 nm was absent , suggesting that the uv exposure 
 was not able to induce much conjugation even though the 400430 
 nm peak was attenuated .
this is consistent with our interpretation 
 of a competition between uv - induced cross - linking and ligand dissociation 
 which limits the extent of cross - linking that can occur .
such a competition 
 was probably not observed in the case of gold , since uv light does not excite gold as it does for semiconductor 
 qds and uv - induced ligand dissociation does not occur for the stable 
 au  s bond .
uv  vis absorption spectra of qd - da in water as 
 a function 
 of exposure to room light over a 2-week period that ( a ) had been and 
 ( b ) had not been exposed to 30 min of uv light prior to transfer to 
 water .
further evidence that uv light 
 leads to only partial cross - linking 
 of qd - da and competing uv - induced dissociation of the da ligands from 
 the qd is found using ft - ir spectroscopy .
figure 3 shows the ft - ir transmittance spectra in three regions of 
 interest as a function of exposure time to uv light . in figure 3a ,
the 31002500 cm region 
 shows the sp c  h stretching peaks and the asymmetric 
 stretch at 2922 cm is used to normalize the spectra . 

the peak 
 at 3012 cm first increased slightly and then decreased 
 upon uv exposure .
this peak is usually assigned to alkenyl c  h 
 peaks from c = c bonds .
however , cross - linking of diacetylenes 
 should not result in such a peak , unless side - reactions occurred that 
 led to incomplete cross - linking
. this may be possible if there was 
 no adjacent diacetylene to cross - link to , due to partial ligand removal , 
 leaving a reactive c = c radical that may have led to the formation 
 of a c = c  h bond .
figure 3b shows the 21002300 cm region as a function 
 of exposure time of the qd - da to uv light , where cc bonds 
 have a weak but isolated absorbance .
in fact , two peaks are observed 
 in this region , one at 2153 cm and another at 
 2254 cm ( black arrows ) , which are characteristic 
 of diacetylenes . after 20 min of uv exposure , 
 the 2254 cm peak effectively disappeared , while 
 the 2153 cm peak diminished only slightly .
as expected from the 
 cross - linking reaction ( scheme 1 ) , the cc 
cc 
 moieties become ( c = c  cc)n , and the change from a double peak to a 
 single peak within 20 min may reflect this .
however , the fact that 
 both peaks decreased within 100210 min suggests that neither 
 type of ligand was present on the particles after extended uv exposure . 

figure 3c highlights the 14501700 cm region , which shows peaks related to c = c and 
 c = o stretching as well as c  h bending modes .
the peaks 
 at 1573 and 1604 cm are assigned as stretches 
 from the c = o group from deprotonated and protonated carboxylic 
 acids , respectively , forming a close - packed hydrogen - bonded network 
 of ligands .
h bending modes , which remained constant within 20 min 
 of uv exposure and reduced in the 20100 min time window . since 
 the c 
h stretching frequency was normalized , this reduction 
 may indicate a significant change in the ligand arrangement on this 
 time scale that partially reduces the oscillator strength of bending 
 modes , which could be the result of the cross - linking process increasing 
 the rigidity of the sam .
a small peak at 1652 cm appearing after 20 min of uv exposure ( black arrow ) , which was not 
 present before cross - linking , may also be indicative of c = c 
 bonds forming during cross - linking , but due to several overlapping 
 peaks in this region , it is difficult to make a definitive assignment 
 at this point .
the ft - ir data can be summarized as follows : changes 
 in the diacetylene peaks show that the uv light caused these groups 
 to react within 20 min and that they are completely gone within 100 
 min .
these changes were also accompanied by small increases in peaks 
 associated with alkenyl c  h and c = c bonds in 20 min 
 that then decreased within 100 min , suggesting side reactions limited 
 the extent of cross - linking . in the 020 min time period , the 
 c = o peaks remained but also started to decrease in the 20100 
 min period . finally , changes in the c 
h bending modes suggest 
 significant ligand rearrangement on this 20100 min time scale . 
 taken together , the ft - ir data supports the fact that diacetylenes 
 can be partially cross - linked on qd - da within 20 min of uv exposure , 
 but more complete cross - linking using uv light was inhibited by the 
 fact that there was a competing process of ligand rearrangement and 
 dissociation that took over on the 20100 min time scale of 
 uv exposure .
the photoluminescence intensity as a function of 
 time exposed to 
 uv and visible light ( sequentially ) is presented in the supporting information , which shows the effect 
 of these partial cross - linking and ligand dissociation processes on 
 the pl properties .
this data provides more evidence that the cross - linking 
 mechanism can be initiated using both uv and visible light and that 
 radicals can actually improve the pl of the qds , in agreement with 
 a recent study , but that continued exposure 
 to uv light is extremely detrimental to the particles . in a previous 
 study
, radicals were generated by external 
 initiators to improve the pl of the qds , but the supplementary pl 
 data here shows that radicals generated by the photoexcitation of 
 qds themselves can have a similar effect .
ft - ir transmittance spectra 
 of qd - da as a function of uv exposure 
 time showing ( a ) c  h stretching , ( b ) cc stretching , 
 and ( c ) c = c and c = o stretching , as well as c 
the significant advantage 
 of using visible - light qd - mediated photocatalysis 
 for cross - linking rather than uv light is highlighted in figure 4 .
photographs of the fluorescence from qd - da with 
 or without 30 min prior exposure to uv light dispersed in water and 
 then exposed to room light were taken over several days . as a comparison , 
 qds solubilized with one of the
most commonly used , non - cross - linking 
 thiolated ligands , qd - mpa , is shown .
qd - mpa showed extensive precipitation 
 from solution even after the first day and the effect was worsened 
 by prior uv exposure , which would be expected for a uv - induced ligand 
 dissociation process .
both the qd - da samples showed 
 significant improvement in colloidal stability over qd - mpa .
however , 
 the non - uv - exposed qd - da showed better long - term stability than the 
 uv - exposed qd - da , highlighted by the fact that the particles remained 
 suspended in solution for the whole time period .
the uv - exposed qd - da 
 began to precipitate from solution within 57 days , and by 
 day 11 was considerably worse than the non - uv - exposed qd - da .
fluorescence 
 photographs under 366 nm ( long wave ) uv light of qd - mpa 
 and qd - da samples that either were or were not exposed to 30 min of 
 254 nm ( short wave ) uv light first to show colloidal stability and 
 fluorescence intensity as a function of time .
a fluorescence layer 
 at the bottom of the vial indicated extensive aggregation of the qds . 

only the non - uv - exposed qd - da samples show no visible aggregation 
 over time .
the degree of aggregation or dispersion 
 in uv - exposed and non - uv - exposed 
 qd - da was then analyzed using single particle fluorescence spectroscopy 
 on a picoquant microtime 200 fluorescence confocal microscope , after being left in aqueous solution for 14 days under room light . 

samples were stirred and diluted to pm concentrations and analyzed 
 using burst integrated fluorescence and fluorescence correlation spectroscopy 
 ( fcs ) , the results of which are shown in figure 5 .
example burst integrated fluorescence traces of particles diffusing 
 through the confocal volume of a fluorescence microscope for ( a ) visible - light - exposed 
 ( black ) and ( b ) uv - light - exposed ( red ) qd - da , obtained after they 
 have been sitting in water for 14 days and ( c ) fcs autocorrelation 
 analysis of each qd - da sample to show average diffusion times of particles 
 through the confocal volume . for the non - uv - exposed qd - da , fitted 
 curves with ( thick black ) and without ( thin black ) taking blinking 
 into account are included , with both fits recovering at the same diffusion 
 times .
blinking is not needed to be taken into account for uv - exposed 
 qd - da ( red ) due to aggregation .
figure 5a shows an example of a burst 
 integrated 
 fluorescence trace from visible - light - exposed qd - da , while figure 5b shows an example from uv - exposed qd - da .
the data 
 is binned into 1 ms time points , with the intensity of each point 
 corresponding to the average detector count rate during that 1 ms 
 time bin .
the peaks in fluorescence intensity arise from fluorescent 
 particles diffusing through the diffraction - limited focused laser 
 beam ; a single peak corresponding to a single particle diffusing through 
 the focus .
the non - uv - exposed samples show much lower intensity bursts , 
 with each peak being narrow , compared to the wide , intense peaks from 
 the uv - exposed samples . these intense ,
wide peaks are due to large , 
 highly fluorescent aggregates of qds slowly diffusing through the 
 laser focus .
the average diffusion time of particles diffusing through 
 the focus is analyzed by calculating the autocorrelation function 
 ( acf ) from the burst integrated fluorescence traces , which is shown 
 in figure 5c .
the black curve shows the acf 
 for the non - uv - exposed qd - da , while the red curve shows uv - exposed 
 qd - da .
the diffusion coefficients are calculated by fitting these 
 curves to extract diffusion times through the confocal volume .
the 
 data were fit to eq 1 , to take blinking into 
 account as previously described.1where2with w0 and z0 describing the width ( 500 nm ) 
 and height ( 2 
 m ) of the confocal beam , f describes the fraction 
 of particles that blinked during the transit time in the beam and a and  describe the power - law blinking dynamics , 
 as previously described . fits both with and 
 without taking blinking into account were performed ( i.e. , either 
 by setting f = 0 or allowing it to be fit ) . for the 
 non - uv - exposed samples ,
the data are fit better at shorter times by 
 taking blinking into account , but both recovered the same diffusion 
 times .
blinking was not a factor for the uv - exposed samples , which 
 is expected if particles have aggregated .
diffusion times ( d ) through the confocal volume of 3.09  0.11 ms for the 
 non - uv - exposed qd - da ( i.e. , cross - linked by qd - mediated visible - light 
 photocatalysis ) and 265  5 ms for the uv - exposed qd - da were 
 found .
these translate into diffusion constants of 20  1 and 
 0.236  0.004 m / s , respectively , at the lab 
 temperature of 20 c . using the stokes 
einstein relation 
 in water at 20 c , these represent hydrodynamic radii of 10 nm 
 for the qd - mediated visible - light cross - linked qd - da and 900 nm for 
 the uv - exposed qd - da . on the basis of the sizes of the core 
shell 
 qd and the ligand , 10 nm is a reasonable hydrodynamic radius to be 
 expected for single particles , while 900 nm highlights the extensive 
 aggregation of the uv - exposed qd - da .
the lack of aggregation of visible - light 
 cross - linked qd - da is also evident from the fact that the blinking 
 parameters are needed to fit the data .
we also measured the fluorescence 
 lifetime of these samples to determine the extent of fluorescence 
 quenching caused by the aggregation ( supporting 
 information ) .
the qd - mediated visible - light photocatalysis 
 resulted in cross - linked qds that show a fluorescence lifetime in 
 water of 9.2 ns , while the uv - exposed qds show a shorter fluorescence 
 lifetime of 6.9 ns .
it is important to reiterate here that no purification 
 step from potential aggregates was performed before taking either 
 of these measurements .
the results from this study are schematically 
 depicted in figure 6 , in which using a qd - mediated 
 visible - light cross - linking approach for the ligands was able to both 
 significantly avoid interparticle cross - linking and avoid ligand dissociation 
 that resulted from uv exposure .
ligand dissociation results in qd 
 aggregation over time , while extensive cross - linking of ligands is 
 able to maintain colloidal stability for long periods of time , highlighting 
 the major outcome from this study .
schematic of the differences observed 
 using our visible - light qd - mediated 
 approach to cross - linking compared to using uv light to directly cross - link 
 the ligands . under the high energy uv light
, ligand dissociation competes 
 with cross - linking that leads to only partial cross - linking of the 
 ligands and eventual aggregation of the qds .
we have designed a novel visible - light photo - cross - linking 
 approach 
 to qd long - term stabilization in aqueous solution .
this was achieved 
 by synthesizing a photo - cross - linkable diacetylene ligand and using 
 the qd to improve the cross - linking of the ligands on the particle 
 by a visible - light photocatalytic mechanism which , in turn , improved 
 the colloidal properties of the same qd that helped cross - link the 
 ligands .
this method combines the advantage of using ligand exchange 
 to render qds water - soluble with the advantage of controlling cross - linking 
 so that it remains intraparticle rather than interparticle , thus avoiding 
 the formation of aggregates and the need for purification .
this new 
 approach has the potential to lead to smaller particle sizes than 
 is possible using amphiphilic polymers by further reducing the size 
 of the bifunctional ligand , although it will be necessary to optimize 
 the correct balance between chain length and sam packing density to 
 achieve such a goal .
furthermore , other water - soluble bioreactive 
 functionalities can be incorporated , since the cross - linking moiety 
 is completely separated from the bioreactive functional group on the 
 particle , and does not rely on any other reagents added to the solution 
 to achieve the cross - linking .
 ligand cross - linking is known to 
 improve the colloidal stability 
 of nanoparticles , particularly in aqueous solutions . 
 however , most 
 cross - linking is performed chemically , in which it is difficult to 
 limit interparticle cross - linking , unless performed at low concentrations . 
 
 photochemical cross - linking is a promising approach but usually requires 
 ultraviolet ( uv ) light to initiate . 
 using such high - energy photons 
 can be harmful to systems in which the ligand  nanoparticle 
 bond is fairly weak , as is the case for the commonly used semiconductor 
 quantum dots ( qds ) . here 
 , we introduce a novel approach to cross - link 
 thiolated ligands on qds by utilizing the photocatalytic activity 
 of qds upon absorbing visible light . 
 we show that using visible light 
 leads to better ligand cross - linking by avoiding the problem of ligand 
 dissociation that occurs upon uv light exposure . 
 once cross - linked , 
 the ligands significantly enhance the colloidal stability of those 
 same qds that facilitated cross - linking . 
Introduction
Experimental Section
Results
and Discussion
Conclusions