in recent years , nanoparticles 
 have been used increasingly for 
 biomedical applications , including drug or gene delivery , imaging , 
 sensing , or photothermal therapy . in particular ,
gold nanoparticles ( nps ) have been suggested as highly 
 useful sensitizing agents in phototherapy due to their unique size 
 and shape - dependent optical properties , high absorption coefficients , 
 ease of synthesis , biocompatibility , and their ability to hold a variety 
 of functional ligands .
it is well - known that citrate - stabilized gold nps 
 are endocytosed by cells and remain in intracellular vesicles .
moreover , targeting of specific sites inside 
 the cell by functionalization of the surface with cell penetrating 
 peptides or peptides containing nuclear localization sequences has 
 been reported . alternatively , to selectively 
 target specific types of cells , gold nps can be modified with suitable 
 antibodies .
this approach has been studied for potential use in photothermal 
 cancer therapy , where cancer cells overexpressing human epidermal 
 growth factor receptor 2 ( her2 ) or epithelial growth factor receptor 
 ( egfr ) were incubated with gold nps conjugated to anti - her2 or anti - egfr antibodies , respectively . 

the nps were then irradiated with light within their plasmon resonance 
 absorbance band to heat the cells to temperatures leading to cell 
 death .
most interestingly , it has also been demonstrated 
 that endocytosis 
 of gold nps by cancer cells and subsequent irradiation of such intracellular 
 nps can lead to cell death even at irradiation levels that are not 
 high enough to cause significant heating .
this nonthermal 
 route to laser - induced cell death has been ascribed to an as yet not 
 fully characterized photochemical reaction , although irradiation of 
 endocytosed nps was reported to be accompanied by increased levels 
 of reactive oxygen species .
the use of such a photochemical 
 mechanism could be of great advantage in situations where different 
 types of cells coexist in close vicinity , since it would allow for 
 more selective targeting of particular cells , whereas  due to 
 the fast diffusion of heat over the relevant length scales 
photothermally 
 induced cell death will affect all cells within the irradiated volume 
 more or less indiscriminately , as long as some of them contain nps .
irradiation of gold nps with continuous wave ( cw ) lamp or laser 
 light has been shown to lead to photogeneration of singlet oxygen 
 ( o2 ) in vitro , suggesting that this highly reactive species , which is widely used 
 in photodynamic therapy , may be involved in 
 the photochemical pathway of cell killing by gold nps . in 
 vitroo2
photogeneration by irradiation 
 of spherical gold nps with short laser pulses or cw 
 laser light at comparable powers and intensities has also been reported . in this study
, it was suggested that the mechanism 
 of singlet oxygen photogeneration may involve  hot  
 electrons , i.e. , the highly excited conduction band electrons which 
 upon absorption of a short laser pulse by a np can reach quasi - equilibrated 
 energy distributions corresponding to temperatures of several thousand 
 degrees .
this seems somewhat surprising , since cw 
 light at the intensities used does not yield  hot 
here we present new experimental results on the photogeneration 
 of singlet oxygen by irradiation of gold nanoparticles with continuous or pulsed laser light , as well as theoretical work pertaining 
 to the underlying mechanism(s ) , which so far had not been addressed . 

we show that electron temperatures in excess of 2000 c are easily 
 achieved in pulsed laser irradiation experiments , whereas cw light 
 under similar conditions yields electronic temperatures of at most 
 10 c above room temperature .
thus , the photogeneration of o2 by gold nps proceeds by different mechanisms 
 under different irradiation conditions ; the implications for the further 
 development of medical applications of the effect are discussed in 
 detail .
furthermore , we also found that even a moderately thick , but 
 dense , ligand layer significantly reduces the efficiency of o2 photogeneration at the np surface , which also has important 
 consequences for practical applications .
citrate - stabilized spherical 
 gold nps with 15 and 46 nm diameter were prepared according to the 
 turkevich  frens and a seeded growth method , respectively .
gold nanorods ( nrods ) were synthesized 
 using the seeded - growth method reported by dickerson et al . with slight modifications . more details of the 
 np preparation are provided in the supporting information .
nps were characterized using uv  vis spectroscopy 
 ( genesys 10 uv ) , differential centrifugal sedimentation ( cps instruments 
 dc24000 ) , and transmission electron microscopy ( tem , fei tecnai spirit 
 microscope at 120 kv ) ( see figures s1 and s2 in the supporting information ) .
the uv  vis absorbance spectrum 
 of nrods showed a transverse plasmon resonance band at 522 nm , and 
 a longitudinal plasmon resonance band at 798 nm and tem revealed nrods 
 to have a length of 40 nm and a diameter of 12 nm .
functionalization 
 with thiolated peg ligands or peptides was achieved by overnight incubation 
 with excess ligand , followed by repeated centrifugation for excess 
 ligand removal .
the capping ligands used here were peg - oh ( hs-(ch2)11-(eg)4-oh ) , mpeg5000 ( hs-(ch2)2-(eg)n - o - ch3 , average mw 5000 g mol ) and peptide c - tat ( primary 
 sequence calnnagrkkrrqrrr ) ; see figure s3 for the structures of the peg ligands .
o2 was 
 detected via the bleaching of 1,3-diphenylisobenzofuran ( dpbf ) , 
 which is widely used for this purpose .
all experiments involving dpbf 
 were carried out in the dark . because dpbf is not soluble in neat 
 water ,
all experiments were conducted 
 in 50/50 ( v / v ) mixtures of water and ethanol .
a fresh solution of 
 dpbf ( 3.1 mg , 0.115 mm , a412  nm = 2 ) in etoh ( 100 ml ) was kept stirring in the dark .
a 10 mm quartz 
 cuvette and a 3 mm stirrer bar were left in aqua regia ( 1:3 hno3:hcl ) for 15 min and thoroughly rinsed multiple times with 
 milli - q water ( mq h2o ) and etoh . in the clean cuvette with 
 stirrer bar , either mq h2o ( 600 l ) or np solution 
 ( 600 l ) was mixed with the ethanolic dye solution ( 600 l ) . 

where appropriate , the np concentration was adjusted prior to mixing 
 to yield an absorbance of 0.4 at 532 nm in the final solution .
the 
 cuvette was sealed with an airtight lid and parafilm , and the uv  vis 
 absorbance spectrum was recorded ( genesys 10 uv ) .
the sample was then 
 placed on a stirring plate ( in the dark ) , and its absorbance spectrum 
 measured every 10 min for 30 min to ensure the solution was stable . for the irradiation experiments ,
the cuvette containing 1200 l 
 of sample solution was placed on a stirring plate in front of the 
 laser . for experiments with nps ,
the cuvette was fitted with heat 
 fins using non - silicone heat transfer paste and cooled using a fan . 

no significant increase of the cuvette temperature beyond a slight 
 warming was observed . for most experiments ,
the sample was irradiated 
 at 1000 mw ( unless stated otherwise ) using a 532 nm continuous - wave 
 diode pumped solid state laser ( laser quantum opus 532 ) with a 1/e beam diameter of 1.85 mm ; for some experiments 
 the beam was expanded to 8 mm diameter using a lens , as stated explicitly . 

the sample was irradiated for 10 min and then removed from the laser 
 setup to record the absorbance spectrum ; this was repeated until the 
 sample had been irradiated for 60 min in total .
irradiation of gold 
 nrods in the longitudinal plasmon resonance band at 800 nm was performed 
 in the same setup , but using a titanium : sapphire laser ( coherent mira 
 900 ) , aligned on the auxiliary cavity , which prevents mode - locking 
 and thus provides continuous - wave laser operation at 800 nm with 1000 
 mw power ; the beam was expanded to 3.4 mm using two lenses . pulsed 
 laser irradiation was performed in the same setup , but using the second 
 harmonic of a q - switched nd : yag laser ( quantel brilliant ) ( 532 nm , 
 5 ns pulse length , 10 hz repetition rate , 3.5 mm beam size , 15 mj 
 pulse energy ) . for experiments that included nps , the absorbance 
 of dpbf at 412 

nm was calculated by subtracting the np absorbance at 412 nm ( obtained 
 from neat np samples ) from the measured sample absorbance .
the concentration 
 of dpbf in the samples always yielded an initial absorbance very close 
 to 1 at 412 nm ; for comparative data analysis , the irradiation - time - dependent 
 dpbf absorbance at 412 nm was therefore normalized to 1 at zero irradiation 
 time .
1,3-diphenylisobenzofuran 
 ( dpbf ) 
 readily undergoes a 1,4-cycloaddition on reaction with o2 to form endoperoxides which irreversibly yield 1,2-dibenzoylbenzene . 

dpbf strongly absorbs light at 412 nm ( figure 1a ) , but due to the loss of the -system 
 of isobenzofuran , the product does not absorb light at this wavelength ; 
 it is this loss of absorbance that is used to detect the presence 
 of singlet oxygen . here
, dpbf was chosen 
 as singlet oxygen sensor as it has no absorbance at 532 or 800 nm , 
 the wavelengths used for laser irradiation of gold nanoparticles in 
 their plasmon resonance bands , and no photobleaching of dpbf was expected 
 to occur upon irradiation in the absence of nps .
contrary to this 
 expectation , some bleaching was observed under our cw irradiation 
 conditions , with the dpbf absorbance decreasing by ca .
10% upon irradiation 
 at 1 w ( 37 w cm ) for 60 min ( figure 1a ) .
as shown in figure 2a , and described in more detail 
 in the supporting information , this photobleaching 
 of dpbf has two phases : ( i ) a rapid phase , extending over the first 
 1020 min of irradiation under the conditions used here , and 
 ( ii ) a slower phase , which on the time scale investigated appears 
 linear with time .
we found that phase i depends on laser power , beam 
 size , and the presence of oxygen , whereas the slope of the time dependent 
 bleaching after 20 min , phase ii , is essentially independent of the 
 presence of oxygen and of the beam power / intensity in the range used 
 here ( see figure s4 for details ) .
it should 
 be noted that similar effects have been reported previously , with 
 cw irradiation at 514 nm for 60 min at significantly lower powers 
 ( 40 mw ) than employed here , leading to a ca .
5% decrease of the absorbance 
 of dpbf in benzene , although no explanation was suggested in that 
 report .
the oxygen dependence of phase 
 i is in agreement with direct photogeneration of singlet oxygen by 
 visible light , which has been suggested to be the reason for dpbf 
 photobleaching upon irradiation with light at wavelengths above 470 
 nm .
however , the slower phase ii is not 
 affected by removing oxygen from the solution ( see figure s4b and figure 3 ) and hence can not be ascribed to singlet oxygen formation . 
 at the present moment , the mechanism of both phases of dpbf photobleaching 
 remains unclear , although both effects are highly reproducible and 
 bleaching does not occur when the sample is not exposed to light ( see figure s4a ) .
photobleaching of the dpbf absorbance 
 upon cw irradiation at 532 
 nm , 1 w ( 37 w cm ) , in a 50/50 ( v / v ) mixture of 
 water and ethanol : ( a ) in the absence of nps and ( b ) in the presence 
 of 15 nm citrate - stabilized spherical gold nps . shown
are absorbance 
 spectra taken at intervals of 10 min from before the irradiation up 
 to a maximum irradiation time of 60 min ; the arrows indicate the direction 
 of change .
( a ) time dependence of the photobleaching of 
 the dpbf absorbance 
 at 412 nm , a412(dpbf ) , upon cw irradiation 
 at 532 nm , 1 w ( 37 w cm ) , in the absence and presence 
 of citrate - stabilized spherical nps with 15 and 46 nm diameter ; shown 
 here are the results from several individual experiments ( dashed lines ) 
 and the average ( solid lines ) , after subtraction of the np absorbance 
 and normalization to 1 at time zero ; see the experimental 
 section for details of data treatment and analysis .
( b ) effect 
 of the nps alone , calculated by subtracting the photobleaching effect 
 of dpbf in the absence of nps from the results obtained in the presence 
 of nps ; the solid lines in ( b ) are linear fits of the data in the 
 range 2060 min .
gradient of the time - dependent dpbf absorbance photobleaching in 
 the irradiation time window 2060 min for different samples 
 under cw irradiation at 532 nm , 1 w ( 37 w cm ) . 
 for experiments in the presence of nps
, the np concentration was adjusted 
 to yield an absorbance of 0.4 at 532 nm .
experiments for dpbf in the 
 absence of nps and in the presence of 15 nm spherical nps were also 
 undertaken after bubbling the sample with nitrogen for 10 min , as 
 indicated ( + n2 ) .
the error bars correspond to the standard deviation 
 of several repeat experiments , and  indicates 
 statistically significant differences with respect to the experiment 
 on dpbf only , as determined by the anova f - test at p < 0.001 ; it should be noted that the results for 15 or 46 nm 
 spherical nps without nitrogen bubbling or a peg capping layer were 
 found to be different to all other results at this statistical significance 
 level .
no repeat experiment was undertaken for irradiation of nanorods 
 at 532 nm , but the same result ( no additional bleaching in the presence 
 of nanorods ) was obtained for irradiation at 800 nm ( figure s5 ) . in the presence 
 of citrate - stabilized spherical gold nps ,
the photobleaching 
 of dpbf upon irradiation at 532 nm , i.e. , within the nanoparticle 
 plasmon resonance band , is significantly increased ( figure 1b ) .
the time dependence of 
 the dpbf photobleaching in the absence of nps and in the presence 
 of nps with 15 and 46 nm diameter is shown in figure 2a .
it can be clearly seen that the 
 presence of nps leads to a significant increase of the dpbf photobleaching 
 effect and that larger nps yield a larger effect although the np absorbance 
 was adjusted to be the same for all samples , so that the same amount 
 of light was absorbed .
the additional photobleaching of dpbf caused 
 by the presence of nps can be clearly ascribed to the generation of 
 reactive oxygen species ( ros ) , since purging of the samples with nitrogen 
 removes this effect ( see below ) .
moreover , previous experiments had 
 shown that light irradiation of citrate - stabilized spherical gold 
 nps leads to the characteristic luminescence of o2 at 1280 nm and does not result 
 in the generation of superoxide , o2 , 
 or hydroxyl , oh , radicals , which leads 
 us to conclude that the predominant ros species produced here is singlet 
 oxygen , o2 . the fact that significant 
 dpbf photobleaching occurs even in the absence of nps requires careful 
 consideration for the analysis of these data .
like dpbf photobleaching 
 in the absence of nps , the effect in the presence of nps shows two 
 phases .
however , subtraction of the effect observed when only the 
 dye is present yields an essentially linear time dependence for the 
 additional effect ascribed to the nps on the time scale of the experiment 
 ( figure 2b ) , indicating 
 that the np - induced photogeneration of o2 is 
 essentially constant over our experimental time interval .
moreover , 
 the second phase of direct dpbf photobleaching is largely independent 
 of the laser power , at least down to 0.1 w , i.e. , significantly lower 
 powers than those used for most of the experiments reported here , 
 as shown in detail in the supporting information ( figure s4a ) , and therefore should be independent of the presence 
 of nps whose absorbance leads to a decrease of the laser power along 
 the beam path , allowing a direct comparison of the results obtained 
 in the time frame of 2060 min .
moreover , this slower phase 
 of direct dpbf photobleaching is also independent of the presence 
 of oxygen . for these reasons , only data starting at 20 min irradiation 
 will be used for quantitative comparisons .
figure 3 summarizes 
 the main results obtained here after np photoexcitation at 532 nm . 

it is very obvious that citrate - stabilized spherical gold nps induce 
 significantly faster dpbf photobleaching than is observed in their 
 absence ( black ) ; this is found for 15 nm gold nanoparticles ( red ) 
 but is even more pronounced for larger nps with 46 nm diameter ( blue ) . 

increasing the size of the spherical nps from 15 to 46 nm increases 
 the np - induced dpbf photobleaching ( i.e. , the additional effect , after 
 subtracting the direct dpbf photobleaching effect ) by ca .
it is interesting to note 
 that unlike the direct dpbf photobleaching , which is not affected 
 by nitrogen purging , the np - induced additional bleaching is significantly 
 reduced by nitrogen purging , almost down to the level of the direct 
 dpbf photobleaching effect .
this strongly supports the conclusion 
 that the np - induced effect is caused by the formation of o2 which then leads to dpbf photobleaching .
furthermore , 
 capping the 15 nm gold nps with peg - oh , a moderately large ligand 
 ( figure s3 ) , essentially removes the np - induced 
 bleaching and reduces the observed effect to the level of the direct 
 dpbf photobleaching .
gold nanorods ( nrods ) with a length of 
 40 nm and a diameter of 
 12 nm , which have two plasmon resonance bands , namely the transverse 
 band at 522 nm and the longitudinal one at 798 nm ( figure s1a ) , stabilized with a capping layer consisting of 
 a mixture of a peg polymer ( mpeg5000 ) and a peptide ( c - tat ) ( see the experimental section ) , were also investigated .
irradiation 
 at 532 nm , i.e. , in the transverse plasmon resonance band , showed 
 no additional dpbf bleaching above the direct dpbf effect ( figure 3 ) .
irradiation with 
 1 w ( 11 w cm ) cw laser power at 800 nm , i.e. , 
 in the longitudinal plasmon resonance band ( figure s5 ) , showed reduced direct dpbf photobleaching compared to 
 irradiation at 532 nm , but again no additional photobleaching was 
 observed in the presence of nrods .
these results show that the nrods 
 used here , which have a ligand layer consisting of a mixture of a 
 peg polymer and a peptide , do not induce the formation of o2 upon laser irradiation , independent of the plasmon 
 resonance which is photoexcited .
irradiation at 532 nm in the 
 presence and absence of citrate - stabilized 
 spherical gold nps with 15 nm diameter was also investigated using 
 laser pulses with a pulse duration of 5 ns ( figure 4 ) . in this case , the uv  vis spectra 
 show a slight broadening of the np plasmon resonance band at 520 nm 
 during the first 5 min of irradiation , suggesting that some aggregation 
 occurs ; after the initial 5 min , however , the nps remain stable .
analysis 
 of the spectra shows that photobleaching of dpbf in the absence of 
 gold nps is similar to the results obtained using cw laser irradiation 
 at comparable average powers ( figure s4a ) . in the presence of gold nps with 15 nm diameter , on the other 
 hand , the np - induced bleaching effect ( i.e.
, the effect remaining 
 after subtracting the direct dpbf photobleaching effect ) which is 
 induced by pulsed laser irradiation is larger than that caused by 
 cw irradiation by almost 1 order of magnitude , in spite of the significantly 
 lower laser power employed during the pulsed irradiation experiments 
 ( 0.15 w vs 1 w ) , which leads to a correspondingly lower number of 
 absorbed photons ; compare figure 4b ( pulsed irradiation ) with figure 3 ( cw irradiation ) .
photobleaching of the 
 dpbf absorbance upon laser irradiation with 
 5 ns laser pulses at 532 nm , 0.15 w , 10 hz repetition rate ( corresponding 
 to a power density of 1.5 w cm and a pulse energy 
 density of 0.15 j cm ) .
( a ) absorbance spectra 
 in the presence of citrate - stabilized spherical gold nps with 15 nm 
 diameter , taken at intervals of 5 min from before the irradiation 
 ( gray ) up to a maximum irradiation time of 30 min ; the arrow indicates 
 the direction of change .
( b ) gradient of the time - dependent dpbf photobleaching 
 ( measured at 412 nm ) in the irradiation time window of 2030 
 min in the absence and presence of nps .
it is straightforward to estimate the quantum yield of np - induced 
 dpbf photobleaching from the absorbed laser power and the observed 
 rate of absorbance bleach .
for the citrate - stabilized spherical nps 
 with 15 and 46 nm diameter under cw irradiation at 532 nm , this yields 
 values of 5  10 and 8  10 , respectively .
thus , less than one of each 1 million photons absorbed 
 by a np leads to the photobleaching of a dpbf molecule under cw irradiation . 
 for irradiation of 15 nm nps with 5 ns laser pulses , on the other 
 hand ,
the quantum yield of np - induced dpbf photobleaching is 3.5  
 10 , i.e. , almost 2 orders of magnitude larger 
 than for cw irradiation , but still very small .
diffusion of 
 oxygen over the lifetime of o2 ( approximately 6 s in 50/50 water / ethanol ) covers a distance larger than the average distance 
 between dpbf molecules at the concentrations used here , and hence 
 it can be concluded that a significant fraction of the photogenerated o2 which escapes from the np surface should be 
 detected .
10 m , which is close to the 
 concentration used here , is sufficient to detect 50% of photogenerated o2 .
thus , the observed 
 low quantum yield of dpbf photobleaching indicates a very low quantum 
 yield of o2 photogeneration by gold nps ( i.e. , 
 number of o2 generated for each photon absorbed ) , 
 having values of the order of ca . 10 for cw irradiation 
 and ca . 10 for irradiation with nanosecond laser 
 pulses .
our results show that irradiation of citrate - stabilized 
 spherical gold nps at 532 nm , i.e. , in their surface plasmon band , 
 with pulsed or cw laser light leads to the production of o2 , detected here by monitoring the bleaching of dpbf 
 absorbance .
however , the rate of o2 production 
 is much larger when using short laser pulses than when using cw light 
 of comparable intensity . whereas ca .
24% of the dye is bleached after 
 only 10 min of irradiation of 15 nm nps with 5 ns laser pulses at 
 an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached over this 
 time by irradiation of the same nps with cw light at significantly 
 higher power ( 1 w ) ( figure 2 ) .
this difference allows one to draw important conclusions 
 on the mechanism of o2 production by irradiation 
 of gold nanoparticles .
irradiation 
 of spherical gold nps at 532 nm leads to the excitation of their plasmon 
 resonance , which can be described as a coherent oscillatory motion 
 of the conduction band electrons ; this oscillation dephases and decays 
 on the sub-100 fs time scale , with only a very small radiative contribution , 
 so that most of the excitation energy is retained as electronic excitation 
 in the form of electron  hole pairs .
since the photon energy 
 is close to the minimum energy required for direct excitation of d - band 
 electrons into the conduction band of gold , a minor contribution of 
 this excitation mechanism can not be ruled out ; however , d - band holes 
 are filled by conduction band electrons on the 10 fs time scale , yielding 
 essentially the same outcome as excitation of the plasmon resonance 
 band .
the excited electrons initially 
 have a nonthermal energy distribution , and often are referred to as 
  primary hot electrons 
, although the concept of temperature 
 does not strictly apply to such a distribution .
they rapidly ( within 
 less than 500 fs ) equilibrate by electron  electron scattering 
 to yield a fermi distribution corresponding to an elevated temperature 
 and can then be referred to as  hot electrons  .
thiol bond dissociation 
 at the surface of gold nps and have been 
 suggested to be responsible for the creation of o2 by irradiation of gold nps with nanosecond laser pulses .
they lose their energy on the time scale of 
 a few picoseconds by interaction with the lattice ( electron  phonon 
 scattering ) with coupling times that are essentially size - independent 
 for nps above 10 nm in diameter , although they strongly depend on 
 the amount of energy deposited due to the temperature - dependent electronic 
 heat capacity .
because the lattice heat capacity 
 is much larger than the electronic heat capacity , this leaves the 
 electrons and the lattice in equilibrium at a temperature which is 
 significantly lower than the initial electronic temperature ; finally , 
 cooling occurs by heat transfer to the solvent and heat diffusion 
 on the time scale of 10 to a few 100 ps , strongly depending on np 
 size .
since the dissipation of 
 the absorbed energy proceeds on the picosecond time scale , excitation 
 with a nanosecond laser pulse yields a highly nonequilibrium situation 
 during the duration of the laser pulse where energy is continuously 
 deposited into the electronic system and at the same time flows through 
 the lattice into the surrounding solvent .
it is therefore not straightforward 
 to predict the temperature of the hot electrons achieved in such experiments . 

we used the  two - temperature model  for the electron 
 and phonon heat baths coupled to finite - element heat transfer 
 and diffusion simulations in the surrounding solvent to estimate this 
 temperature ; details of these simulations are given in the supporting information .
as shown in figure 5 , under our conditions 
 the electrons are expected to reach a temperature of 2100 c , 
 whereas the lattice reaches temperatures of the order of 1400 c 
 and the solvent in the immediate vicinity of the np a temperature 
 of about 900 c .
time - dependent temperatures of the conduction band electrons 
 ( red ) , 
 lattice ( black ) , and first solvent layer ( blue ) , calculated for our 
 experiments using nanosecond - laser pulse excitation ( 15 nm spherical 
 nps in 50/50 etoh / water , 5 ns laser pulses with 0.15 j cm intensity , solid lines ) and for the experiments described in ref ( 24 ) ( 40 nm spherical nps in 
 80/20 etoh / water , 7 ns laser pulses with 0.03 j cm intensity , dashed lines ) using the  two - temperature model  
 for the electron and phonon heat baths , coupled to finite - element 
 heat transfer and diffusion simulations in the surrounding solvent 
 ( see supporting information for details ) ; 
 time zero corresponds to the center of the laser pulse .
a lattice temperature of 1400 c , which is 
 above the melting 
 temperature for solid gold , albeit present for only a few nanoseconds , 
 might be sufficient to cause temporary melting of the nanoparticle , although the pulse intensities used here are 
 still below the reported threshold for size reduction of gold nps 
 of 15 nm diameter by nanosecond laser pulses .
nevertheless , a minor effect on the shape and/or size of the nps , 
 especially at the upper end of the size distribution , can not be ruled 
 out and may be related to the minor np spectral changes observed upon 
 irradiation which suggest some aggregation to occur ( see above ) . because 
 of the slower heat dissipation around larger nps , these are heated 
 to higher temperatures and hence are more likely to fragment , in agreement 
 with experimental results on nanosecond - laser pulse induced fragmentation , 
 which also show that fragmentation is finished after 5 min under conditions 
 similar to the ones used here . since 
 our nps were prepared without excess citrate , the resulting increase 
 in the ratio of surface area to volume may indeed cause some aggregation . 

it also can not be ruled out that the solvent near the np surface temporarily 
 forms bubbles , although the pulse intensities 
 used here are still below the reported threshold for bubble formation 
 by nanosecond pulses for 15 nm nps .
such 
 bubbles could lead to better thermal insulation , thus potentially 
 increasing the maximum temperatures , but on the other hand , they might 
 prevent oxygen from reaching the np surface , thus reducing the chance 
 of o2 formation .
the formation of o2 from the triplet ground state by interaction with a 
 photoexcited sensitizer requires a change of the electron spin and 
 hence can not result from dipole 
dipole ( frster ) interaction , 
 but only from dexter - type electron exchange coupling ; the latter can 
 be described as simultaneous transfers of an electron from one of 
 the 2 * molecular orbitals on oxygen to a photogenerated hole 
 on the sensitizer and of an electron with opposite spin from a high - energy 
 excited sensitizer level to the same or the other 2 * orbital , 
 resulting in the formation of the  or  singlet oxygen state , respectively .
this mechanism requires significant overlap of the relevant electronic 
 wave functions and hence only occurs at short distances of at most 
 10  . since the photoexcited hot electrons have such a short 
 lifetime , this reaction can only occur if an oxygen molecule happens 
 to be in the vicinity of the np or is temporarily adsorbed to its 
 surface at the moment of excitation .
a similar reaction occurs on 
 photoexcited si nanocrystals , although in this case 
 the reaction is more efficient than for au nps due to the much longer 
 lifetime of the photoexcited excitons in si . thus , the short lifetime 
 of hot electron excitation in gold nanoparticles easily explains the 
 low quantum yield of o2 photogeneration which 
 is observed here .
the excitation of electrons to temperatures 
 exceeding 2000 c means that a significant number have sufficient 
 energy to excite an oxygen molecule to the  state , 
 which has an energy of 0.98 ev above the  ground 
 state ( see figure 6 ) .
the number of hot electrons available 
 at the higher energy  and the number of holes available at the 
 lower energy involved in this two - electron exchange reaction  can 
 be estimated from the density of states of gold and the fermi distribution , 
 as described in more detail in the supporting information .
for example , for a spherical np with 15 nm diameter 
 at an electron temperature of 2100 c , there are 260 electrons 
 within an energy interval of 0.1 ev around the  
 state .
( it should be noted that the relevant energy interval is the 
 width of this state for an oxygen in the vicinity or temporarily adsorbed 
 onto a np , which is not known , so only relative numbers will be used 
 here . )
thus , the electron temperatures achieved in our experiment 
 are sufficient for a significant population of hot electrons and holes 
 at the relevant levels .
schematic diagram showing the population probability f(e ) for a np electron state at energy e near the fermi level , ef , 
 under different 
 conditions : ( a ) in equilibrium at room temperature , ( b ) at an electron 
 temperature of te = 2100 c after 
 electron  electron equilibration (  hot electrons  ) , 
 and ( c ) immediately after the absorption of photons by single electrons 
 (  primary hot electrons  , with population changes highly 
 exaggerated to make them visible ) . also shown are the energies of 
 the ground - state triplet (  ) and lowest - excited 
 singlet state (  ) of oxygen as well as the next 
 singlet state (  ) under the assumption that ef is equidistant from the  
 and  energies .
excitation of an oxygen molecule 
 to o2 requires the simultaneous transfer of 
 an electron from the oxygen to a hole at the energy of the  state and of a hot electron with the opposite spin and an 
 energy at the  ( or  ) level to 
 the oxygen molecule .
the large number of equilibrated hot electrons available 
 during 
 a nanosecond - laser pulse also rules out the primary hot electrons 
 as the main source of o2 photogeneration .
the 
 number of primary hot electrons available during the pulse duration 
 can be estimated from the number of photons which are absorbed per 
 nanosecond , multiplied by their lifetime , which is less than 500 fs .
however , these primary hot electrons populate 
 np states at energies from the fermi level ef to ef + 2.34 ev ( the energy of 
 a photon at 532 nm ) ( see figure 6c ) . for 15 nm nps and excitation with 5 ns laser pulses 
 with 0.15 j cm intensity
, this predicts that not 
 more than three primary hot electrons are available within an energy 
 interval of 0.1 ev around the  level at any time 
 during the laser pulse , assuming that all levels are equally populated 
 and taking into account that the density of states for gold has an 
 essentially constant value in the relevant energy range around ef .
this is significantly 
 less than the number of hot ( equilibrated ) electrons available at 
 the maximum electron temperature , which was estimated to be on the 
 order of 260 for the same energy interval ( see above ) .
thus , we can 
 conclude that it is indeed the hot electrons which are responsible 
 for the observed o2 photogeneration when using 
 short laser pulses for excitation .
similar results to those 
 obtained here using pulsed irradiation 
 have recently been reported for spherical gold nps with 40 nm diameter . although a significantly lower laser pulse energy 
 density ( 0.03 j cm , compared to 0.15 j cm here )
was used under otherwise similar experimental 
 conditions , slightly faster o2 photogeneration 
 was observed in this study , with the dpbf absorbance decreasing by 
 ca .
24% decrease 
 observed here in the first 10 min ( figure 4a ) . in this context , it is interesting to 
 note that in spite of the lower laser pulse energy density the nps 
 are heated to almost the same electron temperature as in our experiments 
 ( see figure 5 ) .
this 
 is largely due to ( i ) heat dissipation from larger nps being slower and ( ii ) the use of 80/20 etoh / water as solvent 
 in ref ( 24 ) , which 
 has slower heat transport than a 50/50 etoh / water mixture .
an explicit 
 calculation of the number of hot electrons available at the energy 
 of the oxygen  state for the two experiments is 
 given in the supporting information ; together 
 with a detailed consideration of all other experimental differences , 
 these numbers yield very good agreement between the expected and the 
 observed relative o2 photogeneration rates 
 ( see the supporting information for details ) . 

this provides further support for the conclusion that o2 photogeneration is mediated by the equilibrated hot 
 electrons of gold nanoparticles under nanosecond - pulsed laser irradiation .
compared to the 
 effect of pulsed irradiation , the rate of o2 photogeneration is much smaller when using cw light of comparable 
 intensity . whereas ca .
24% of the dye is bleached after only 10 min 
 of irradiation of spherical nps with 15 nm diameter with 5 ns laser 
 pulses at an average power of 150 mw ( figure 4 ) , only 12% of the dye is bleached 
 over this time by irradiation of the same nps with cw light at significantly 
 higher power ( 1 w ) ( figure 2 ) . in a previous publication , significantly higher rates of dpbf photobleaching had been reported 
 under conditions which appear to be similar to the ones used here . 
 in this context
, we note that we also observed such significantly 
 higher rates of bleaching , but only when the sample preparation protocol 
 described in the experimental section was 
 not followed accurately ; for example , the use of dye solution that 
 had not been freshly prepared or of a cuvette that had not been cleaned 
 thoroughly and rinsed multiple times with mq water or the use of a 
 cell that was not sealed during irradiation , leading to some loss 
 of ethanol from the solution , all resulted in larger and highly irreproducible 
 bleaching of dpbf under cw irradiation , up to levels comparable to 
 those reported in ref ( 24 ) , even in the absence of nps . in the following
, we will show 
 that photogeneration of o2 under cw irradiation , 
 unlike pulsed irradiation , is mediated by the initially created 
primary 
 hot  electrons ; i.e. , it occurs during the short time during 
 which the excited electrons have not yet relaxed to a thermal distribution 
 ( see figure 6c ) . under 
 the cw irradiation conditions used here ( 1 w , 1.85 mm beam diameter ) , 
 a spherical np with 15 nm diameter absorbs photons at an average rate 
 of 1.3  10 s , as estimated from 
 the absorption cross section and the 
 beam intensity .
this means that after absorption of a photon there 
 is enough time for full relaxation and transfer of the photon energy 
 into the solvent , which occurs in less than 100 ps , before absorption of the next photon .
absorption of one 
 photon by a 15 nm np yields  hot  electrons at a temperature 
 of 10 k above the surrounding after electron  electron equilibration ; 
 these  hot  electrons lose their energy by electron 
phonon 
 scattering within a few picoseconds to yield a np whose temperature 
 is only 80 mk above the surrounding . neither of these effects is expected 
 to yield any significant photochemical effects ; estimates analogous 
 to those described above predict that for a spherical 15 nm np with 
 an electron temperature of 35 c there are ca .
2.6  10 electrons 
 within an energy interval of 0.1 ev around the oxygen  state and the same number of holes around the  state energy .
this means that there are on the order of 10 times less hot electrons and 10 times less holes 
 available for the photoreaction than under our pulsed laser irradiation 
 conditions , which rules out any significant reaction ; this is also 
 confirmed by the fact that these population numbers are less than 
 a factor 2 larger than those for room temperature , where no o2 is generated in the absence of light .
this leaves 
 only the primary hot electrons , i.e. , those electrons 
 that are excited upon absorption of a photon but have not yet equilibrated 
 by electron  electron scattering , as potential cause for photogeneration 
 of o2 .
absorption of a single photon can potentially 
 excite electrons to energies of up to 2.34 ev above the fermi level . 

if one assumes excitation of only one electron by each photon and 
 equal excitation probability for all available electrons , as shown 
 in figure 6c , on average 
 there will be 0.043 electrons within an energy interval of 0.1 ev 
 around the oxygen  state , which is approximately 
 5000 times less than during a single laser pulse in the pulsed experiments , 
 see above .
taking into account that the same factor also applies to 
 the holes required for the dexter mechanism , but correcting for the 
 lifetime of the excitation ( 500 fs for primary hot electrons created 
 during cw irradiation , ca .
3 ns for the hot electron distribution induced 
 by a single laser pulse ; see figure s6 ) 
 and the repetition rates ( 1.3  10 s for single photon absorption during cw irradiation , 10 s for the pulsed laser irradiation ) , one would predict a rate of o2 photogeneration under our cw irradiation conditions 
 which is smaller than that expected for our pulsed irradiation conditions 
 by a factor on the order of 10 .
this is significantly 
 closer to the experimental results ( ratio of o2 photogeneration rates under pulsed vs cw irradiation of 1020 ) 
 than any estimate based on the equilibrated  hot  electrons 
 after the absorption of a single photon .
the main discrepancy between 
 the predicted and observed cw results arises from the assumption of 
 direct excitation of single electrons in the above estimate , which 
 is not valid for irradiation at 532 nm , i.e. , in the gold np plasmon 
 resonance band , since this leads to the coherent excitation of many 
 electrons which rapidly dephases without the electrons exchanging 
 energy .
thus , absorption of a single photon yields more than one primary 
 hot electron , with the photon s energy distributed over all 
 of them . consequently
, the energy distribution even of the primary 
 hot electrons will not extend up to 2.34 ev above ef but will be shifted toward the states nearer the fermi 
 level , thus increasing the population of states around the oxygen  energy and hence the yield of o2 .
a more quantitative estimate of this effect is beyond the scope 
 of this paper .
an alternative possibility for the mechanism 
 of o2 photogeneration by gold nps could be 
 envisaged , which is 
 based on increased direct photoexcitation of oxygen due to the well - known 
 local electric field enhancement in the vicinity of metal nps by the 
 plasmon electrons .
however , significant field enhancement extends 
 to distances comparable to the dimensions of the nanoparticle , so that this mechanism is in disagreement with 
 our observation that a peg - oh capping layer , which has a thickness 
 of only 2 nm , completely inhibits o2 photogeneration ( figure 3 ) .
dexter - type electron exchange coupling , 
 on the other hand , is known to be of significance only over distances 
 of less than 1 nm , as discussed above , and thus is further supported 
 by this observation . in conclusion ,
cw irradiation is less efficient 
 than pulsed laser 
 irradiation in photogenerating o2 since the 
 ( equilibrated ) hot electrons , which are the main mediator of the photochemistry 
 in the case of pulsed irradiation , do not have sufficient energy / temperature 
 to drive o2 photogeneration .
nevertheless , 
 our results confirm that cw irradiation of spherical gold nps produces 
 detectable amounts of o2 .
the absence of significant amounts of ( equilibrated ) hot electrons 
 means that o2 photogeneration proceeds via 
 a different mechanism under cw irradiation compared to pulsed laser 
 generation ; the above estimates indicate that it is the primary hot 
 electrons , i.e. , the directly photoexcited electrons , which are responsible 
 for the photochemistry here and that the photochemical reaction must 
 occur before these equilibrate by electron  electron thermalization .
photodynamic 
 therapy holds great promises for medical applications , such as the 
 treatment of cancer , because of the ability to selectively affect 
 diseased tissue only .
however , wider use of photodynamic 
 cancer therapy is currently prevented by several limitations imposed 
 by the available photosensitizers .
these limitations include toxicity , 
 poor stability and photostability , poor selectivity for cancer tissue , 
 and the need of using visible light with poor tissue penetration . 

all of these limitations , in principle , can be overcome by the use 
 of gold nanoparticles , which are nontoxic , have excellent stability 
 even under irradiation , can target cancer tissue either passively 
 by the enhanced penetration and retention ( epr ) effect or by active targeting , have extinction coefficients 
 that are larger than those of dye molecules by several orders of magnitude , 
 and can be tuned to absorb in the near - infrared spectral region for 
 maximum tissue penetration .
gold nanoparticles have been reported 
 to have three potential modes of operation for inducing cell death 
 by irradiation , namely ( i ) hyperthermia , which is based on the rapid 
 conversion of the absorbed light energy into heat , ( ii ) np - assisted 
 photodynamic therapy , in which the efficiency of a standard sensitizer 
 is amplified by the np plasmon field enhancement effect , or ( iii ) 
 a direct photochemical mechanism without involvement of a photosensitizer .
the feasibility of photothermal therapy has been clearly established 
 by careful experiments in vitro(1113,15,54 ) and has been shown 
 to work in vivo .
however , it should be noted that most studies reporting successful 
 photothermal therapy made no attempt to either confirm significant 
 heating or rule out photochemical effects , which means that some of 
 these reported results could in fact arise from photochemical rather 
 than photothermal effects or from a synergistic combination of photochemical 
 and photothermal effects . as has been pointed out recently , another problem with many reports on photoinduced 
 hyperthermia using nps is the relatively high light intensity required 
 to reach sufficient temperatures , which often were well above the 
 generally accepted skin tolerance threshold .
the presence of 
 gold nps can also lead to increased photogeneration 
 of o2 by traditional photodynamic sensitizers , which can be ascribed to the local electric field enhancement in 
 the vicinity of metal nps by the plasmon electrons , similar to the 
 well - studied sers ( surface - enhanced raman spectroscopy ) effect .
this 
 approach might help to alleviate some of the drawbacks of traditional 
 photodynamic therapy but does not directly overcome them , since it 
 still requires the presence of a sensitizer .
in fact , the requirement 
 for two active components , gold np and sensitizer , which must be colocalized , 
 introduces an additional complication , and care must be taken to avoid 
 a reduction of the photosensitizer effect due to quenching of its 
 excited state by the metal np . in an alternative approach
, cell death has been demonstrated to 
 occur following irradiation of intracellular ( endocytosed ) gold nps 
 even at irradiation levels that are not high enough to cause significant 
 heating .
this photochemical effect has been related to the 
 observation that irradiation of nps in vitro results 
 in the formation of singlet oxygen , which is the active species in traditional 
 photodynamic therapy .
because of the short lifetime of o2 ( 3.4 s in water ) , its action is highly localized  an oxygen molecule only 
 diffuses over the length scale of 100 
 nm in this time . for this reason , only intracellular nps
are expected 
 to trigger cell death by the photochemical route , although they may 
 initially be located inside endosomes which are known to be broken 
 up by nps under cw irradiation . on the 
 other hand
, the localization of the photochemical effects within individual 
 cells also means that in situations where different types of cells 
 coexist in close vicinity , selective targeting of particular cells 
 and minimization of collateral damage should be achievable .
this is 
 different than photothermally induced cell death , which affects all 
 cells within the irradiated volume more or less indiscriminately , 
 as long as some of them contain nps , because of the fast diffusion 
 of heat over the relevant length scales .
the results of the 
 experiments described here provide more insight 
 into the direct photochemical mechanism and allow some important conclusions 
 to be made for the further development of practical applications . 

they show that detectable amounts of o2 are 
 generated by irradiation of nps with short laser pulses or cw light 
 even in the absence of a photodynamic sensitizer , albeit with low 
 quantum yield .
short laser pulses are significantly more efficient 
 at this process , since they can heat a significant fraction of the 
 np conduction band electrons to high enough temperatures to excite 
 oxygen to the singlet state .
however , this requires pulse energy densities 
 that are well above generally accepted safe levels for the irradiation 
 of skin with pulsed laser light . since the effect requires the absorption 
 of many photons by a np during one laser pulse , it is highly nonlinear 
 with respect to irradiation intensity , and thus it will not be possible 
 to compensate lower irradiation levels by longer irradiation times . 

furthermore , pulsed irradiation at the required intensities also can 
 cause other effects , such as np fragmentation or bubble formation , and it is not clear what consequences 
 these effects may have when occurring in tissue .
although the use 
 of femtosecond laser pulses , as compared to the nanosecond pulses 
 used here , might alleviate some of these problems to some extent , 
 they would require even more sophisticated equipment which may not 
 be suitable for a clinical environment .
the use of cw light , 
 on the other hand , is straightforward and 
 does not even require a laser but can be achieved with simple lamps . 
 in spite of the lower quantum yield of o2 photogeneration 
 by nps under cw irradiation which
is reported here , cell death induced 
 by cw light in the presence of nps has been reported in vitro and in vivo .
although the experiments 
 described here , which did not involve any biological material , were 
 undertaken at light intensities that are above safe irradiation levels , 
 the mechanism of o2 photogeneration by nps 
 under cw illumination is shown to be based on the absorption of single 
 photons .
this means that lower light intensities can be compensated 
 for by longer irradiation times , allowing one to reduce the intensity 
 to safe levels for biological or medical applications without affecting 
 the amount of o2 generated .
o2 photogeneration and cancer cell destruction in vitro and in vivo have indeed been observed upon irradiation 
 on the minute time scale at intensities below safe levels and using 
 nonlaser light sources .
the results presented here indicate another design criterion 
 which 
 needs to be fulfilled for successful implementation of the photochemical 
 route of inducing cell death by irradiation of nps , namely that the 
 nps must not possess a dense capping layer . even the thin capping 
 layer formed by peg - oh , with a thickness of only 2 nm , is sufficient to completely inhibit o2 photogeneration ( figure 3 ) .
this is in full agreement with the suggestion that 
 energy exchange between the np and the oxygen molecule occurs via 
 the dexter ( two electron exchange ) mechanism , which is limited to 
 distances of less than 1 nm . similar effects have been reported for o2 photogeneration by si nanocrystals , which is 
 significantly affected by a thin oxide layer .
it is important to point out that the assay used here for o2 detection ( bleaching of dpbf ) only reports on singlet 
 oxygen found outside the np capping layer ; thus , it can not be ruled 
 out that even on nps with a peg - oh capping layer some o2 is photogenerated at the np surface but reacts with 
 the np ligands and hence is quenched .
on the other hand , such singlet 
 oxygen would not be of any direct use for practical applications , 
 such as the induction of cell death , so that the assay results in 
 fact report the effects relevant for such applications .
uncapped ( citrate ) 
 nps are rapidly covered by a protein corona after they have been taken 
 up into live cells , but it appears that 
 this corona is permeable enough for oxygen to not completely prevent 
 singlet oxygen formation , as evidenced by fact that cell death by 
 the photochemical route has been observed with such nps ; it may be that because of their size proteins 
 are not able to form a capping layer ( corona ) of similar density as 
 the smaller ligands used here .
similarly , a lipid bilayer appears 
 to allow oxygen access to the surface of gold nanorods , whereas a dense pentapeptide ( calnn ) capping 
 layer on spherical gold nanoparticles is sufficient to suppress the 
 photochemical mechanism of cell death .
it also seems likely that the thick peg capping layer present 
 on 
 the nanorods used here is the main reason for the absence of o2 photogeneration by nanorods upon irradiation 
 in either the transversal or the longitudinal plasmon resonance band 
 that was observed here . because of their longitudinal plasmon resonance 
 band , which is in the near - ir spectral region with high tissue penetration , 
 nanorods are more suitable for practical photodynamic applications 
 in tissue .
however , standard synthesis protocols yield nanorods within 
 a bilayer of cytotoxic ctab , so that ligand exchange is required before 
 any biological or medical application , and care will need to be taken 
 to choose suitable ligands to allow access of oxygen to the nanorod 
 surface . potential examples for these are poly(vinylpyrrolidone ) , 
 lipid bilayers , or mesoporous silica , all of which have been used 
 successfully in experiments showing photochemically induced cell death 
 using gold nanorods .
dpbf has been used successfully to detect 
 singlet oxygen that is 
 formed when spherical nps are irradiated at 532 nm , either with cw 
 or pulsed laser irradiation .
singlet oxygen generation by pulsed laser 
 irradiation has been shown to act via the equilibrated hot electrons 
 that can reach temperatures of several thousand degrees during the 
 laser pulse ; cw irradiation , on the other hand , can act only via the 
 directly excited primary  hot  electrons , which rapidly 
 lose their energy by electron 
electron equilibration , and hence 
 is significantly less efficient for the formation of singlet oxygen . 

nevertheless , even cw irradiation can produce enough singlet oxygen 
 for photodynamic therapy applications and will allow practical applications 
 of the effect at safe irradiation levels .
photodynamic therapy using 
 gold nanoparticles will also require careful design of the nanoparticles 
 with respect to size , shape , and capping layer and will require internalization 
 of the nps , not just attachment to the cell surface , which is sufficient 
 for photothermal therapy .
 the 
 formation of singlet oxygen by irradiation of gold nanoparticles 
 in their plasmon resonance band with continuous or pulsed laser light 
 has been investigated . 
 citrate - stabilized nanoparticles were found 
 to facilitate the photogeneration of singlet oxygen , albeit with low 
 quantum yield . 
 the reaction caused by pulsed laser irradiation makes 
 use of the equilibrated hot electrons that can reach temperatures 
 of several thousand degrees during the laser pulse . 
 although less 
 efficient , continuous irradiation , which acts via the short - lived 
 directly excited primary  hot  electrons only , can produce 
 enough singlet oxygen for photodynamic cancer therapy and has significant 
 advantages for practical applications . 
 however , careful design of 
 the nanoparticles is needed , since even a moderately thick capping 
 layer can completely inhibit singlet oxygen formation . 
 moreover , the 
 efficiency of the process also depends on the nanoparticle size . 
Introduction
Experimental Section
Results and Discussion
Conclusions