interest 
 in exploiting nanocrystalline semiconductor quantum dots 
 ( qds ) for a wide variety of disparate applications continues to grow 
 nearly unabated . this interest arises primarily from the unique photophysical 
 properties of qds : high quantum yields ; good photostability and resistance 
 to chemical degradation ; large one- and two - photon absorption coefficients 
 across a broad range of wavelengths ; and narrow photoluminescence 
 ( pl ) that can be tuned as a function of both semiconductor material 
 and core size . these properties have already 
 found extensive use in optoelectronic research , such as for solar 
 energy conversion , and as biological probes 
 for imaging or sensing . considering the latter , qds have 
 been shown to be particularly useful for designing a variety of active 
 biosensing assemblies that rely on changes in energy transfer rates 
 as the mechanism of signal transduction . the most prominent and successful 
 of these configurations are based on frster resonance energy 
 transfer ( fret ) . here , the qd is paired with a suitable donor ( d ) 
 or acceptor ( a ) , and the proximity needed for fret is achieved through 
 a biomolecular linkage such as a protein , peptide , or oligonucleotide . transduction of biological processes such as binding events or enzyme - catalyzed 
 hydrolysis exploits d  a association or dissociation as mediated 
 by the responsive biomolecular linkage located between the d and a. 
 the process of fret with qds is sufficiently understood that on / off 
 sensing schemes can be designed for many biological processes or analytes 
 of interest . as a consequence , other qd 
 energy transfer mechanisms are only partially characterized and remain 
 underutilized , although they may also have much to offer for biosensing . in contrast to many organic 
 fluorophores , qds have significant 
 surface area and , due to their reversible redox properties , are very 
 sensitive to charge transfer processes with surface adsorbates . recent research has focused on harnessing charge transfer quenching 
 of qds for biosensing in a capacity similar to fret . charge transfer is thought to offer several advantages over fret : 
 a putative exponential dependence on d  a separation ( cf . inverse 
 sixth power for fret ) ; no requirement 
 for underlying spectral overlap ; and 
 potentially greater sensitivity to the medium between d and a. consequently , 
 the charge transfer interactions between qds and a variety of redox - active 
 molecules , such as catechols , are being actively studied in different 
 biosensing formats . redox - active metal complexes are also particularly promising as 
 charge transfer partners for qds as they display many attributes that 
 can benefit this role : well - understood redox properties that are extensively 
 documented in the literature , including tuning of those properties 
 through coordinating ligands , and established synthetic chemistry 
 that uses commercially available precursors , offers high yields , and 
 can include appended reactive chemical  handles  for 
 labeling biomolecules . although various 
 complexes of iridium , rhenium , osmium , and iron are commonly used 
 in such roles , it is a ruthenium(ii)phenanthroline ( ru - phen ; see figure 1d ) that has thus 
 far found extensive use as a charge transfer quencher for qds in biosensing 
 applications . ( a ) illustration of the cdse / zns qd and 
 ru - phen - labeled 
 peptide conjugates ; ( b ) dhla - peg ligand solubilizing the qds ; ( c ) 
 modular design of peptide sequence ; and ( d ) structure of ru - phen maleimide used to label the peptide linker . to develop charge - transfer - based qd biosensors , the benson group 
 began by site - specifically labeling intestinal fatty acid binding 
 protein , maltose - binding protein , a designer pb - binding 
 protein , and a thrombin - binding dna aptamer with ru - phen . these labeled proteins were then conjugated to qds , and extensive 
 experimental investigation with appropriate target molecules demonstrated 
 that they could be effective charge transfer sensors for fatty acids , 
 maltose , lead , and thrombin , respectively . within these composite ru - phen  qd 
 assemblies , it was postulated that concentration - dependent analyte 
 binding in the sensor complex induced structural rearrangements that 
 altered the ru - phen position relative to the qd or , in 
 the special case of the fatty acid binding protein , changes in the 
 solvation environment . in turn , these effects resulted in a change 
 in the rate of charge transfer between the qd and ru - phen 
 and thus the qd pl intensity . similarly , initial work from our 
 group has demonstrated that the 
 rate of charge transfer quenching of qd pl depends on the number of 
 ru - phen bound in proximity to the qd . this result formed the basis for a protease sensor that 
 signaled the hydrolysis of peptide linkages between the qd and ru - phen using the recovery of qd pl . subsequent work demonstrated that the assembly of ru - phen - labeled peptides to eight different colors ( i.e. , sizes ) of 
 qd could create a dense , spectral - multiplexing system across the visible 
 spectrum  something that would not be possible with a single 
 so - called dark quenching acceptor and reliance on fret . all of these studies serve to highlight the 
 strong potential that qd  ru - phen interactions 
 have for developing new types of biosensors ; however , this proof - of - concept 
 work is primarily empirical , and a detailed understanding of charge 
 transfer quenching mechanisms in these complex qd  biomolecular  nanocrystal 
 composite materials still remains to be achieved . as such , qd biosensors 
 based on charge transfer quenching are currently designed without 
 the predictive and rational design capabilities associated with fret - based 
 configurations . in this work , we evaluate the underlying electrochemistry 
 and excited 
 state dynamics between aqueous cdse / zns core  shell qds and 
 ru - phen coupled through a peptide linker ( see figure 1 ) . a previous study of this configuration was limited 
 to characterizing the quenching of qd pl intensity and changes in 
 the pl excited state lifetimes on the nanosecond time scale . although useful for demonstrating the overall 
 effect of assembling ru - phen with qds , these experiments 
 offered limited mechanistic insight . while ultrafast spectroscopy 
 techniques have been used in more detailed physical studies of charge 
 transfer with qds , these prior studies have been primarily nonbiological 
 in nature , emphasizing energy conversion instead of sensing applications . 
 in these instances , the redox active moiety has almost always been 
 physisorbed directly to the surface of a core - only , ligand - coated 
 qd , more oftentimes in organic rather than aqueous solvent . to gain 
 further insight into a biologically relevant configuration , we used 
 femtosecond transient absorption spectroscopy ( fsta ) with steady - state 
 and time - resolved pl spectroscopy to study the aqueous charge transfer 
 quenching of core / shell qds by peptide - linked ru - phen . in particular , we examine the charge carrier dynamics and directionality 
 that lead to the quenching of qd pl when ru - phen - labeled 
 peptide is assembled to the qd . overall , we find that qd quenching 
 requires the ru oxidation state , consistent with a charge 
 transfer mechanism , and that shell thickness around the core qd is 
 a significant determinant of quenching efficiency . the fsta data also 
 show that exciton quenching is a rather slow process , consistent with 
 other qd conjugate materials that undergo hole transfer . shell 
 qds with peak pl at 550 and 580 nm ( qd550 and qd580 ) were synthesized 
 as described previously and made water - soluble 
 with a poly(ethylene glycol ) ( peg ; mw 750 da ) appended derivative 
 of dihydrolipoic acid ( dhla ) terminating in a methoxyl group ( dhla - peg , 
 see figure 1b ) . a maleimide derivative of ruthenium(ii)phenanthroline ( ru - phen ) was synthesized as described previously . all experiments were done in phosphate - buffered 
 saline ( pbs ; 10 mm phosphate , 137 mm nacl , 3 mm kcl , ph 7.4 ) . the peptides used 
 in this study are listed in table 1 and were 
 synthesized using standard in situ neutralization cycles with boc - solid - phase 
 peptide synthesis ( boc - spps ) . peptides were labeled 
 with ru - phen maleimide , purified , desalted , quantitated , 
 and lyophilized for storage following a protocol described in detail 
 elsewhere . 1 mg of peptide 
 was dissolved in 1 ml of pbs and mixed with ca . the reaction was agitated for 23 
 h at 2225 c , then overnight at 4 c . labeled peptide 
 was purified over nickel(ii)-nitrilotriacetic acid ( ni - nta)-agarose ( qiagen , valencia , ca ) and desalted using a reverse - phase 
 oligonucleotide purification cartridge ( opc ; applied biosystems ) with 
 triethylamine acetate buffer . purified peptide was quantitated by 
 uv  visible absorption spectrophotometery ( 490 nm = 5000 m cm for ru - phen ) , aliquoted , dried in vacuo , and stored at 20 c 
 until needed . ceric ammonium nitrate , ( nh4)2ce(no3)6 , was dissolved in water to 100 mm . approximately 15 l of this solution ( 15 mol ) was added 
 to a 300 l solution of 100 m p1(ru ) ( 30 
 nmol ) in water . the solution quickly changed from red / brown to colorless , 
 indicating conversion to p1(ru ) . the solution was left 
 at room temperature for 10 min before adding 300 l of 200 mm 
 phosphate buffer ( 60 mol phosphate ) , upon which the solution 
 became cloudy . this mixture was centrifuged at 5000 rcf for 2 min 
 to give a yellow - brown pellet . the precipitate consisted of a mixture of 
 highly insoluble cepo4 and ce3(po4)4 , which have pksp values 
 > 25 . thus , addition of phosphate was a convenient means 
 to remove to the ce from the peptide solution to avoid 
 its potential oxidative effects upon peptide assembly with the qds . while ceric ammonium nitrate is known to oxidize tryptophan and tyrosine , these residues were absent from the peptides 
 in table 1 , and thus no modifications of the 
 remaining amino acid residues were expected . gel electrophoresis was 
 used to confirm that the p1(ru ) still self - assembled 
 to the qds ( data not shown ) . ground state absorption measurements were made using an agilent 
 8453 diode array spectrophotometer and quartz cuvette with a 1.00 
 cm path length . pl intensity measurements were acquired with peptide - linked 
 qd  ru - phen conjugates at a concentration of 0.18 
 m using a tecan infinite m1000 dual monochromator multifunction 
 plate reader equipped with a xenon flash lamp ( tecan , research triangle 
 park , nc ) . the excitation wavelength used was 400 nm , corresponding 
 to an absorption minimum for ru - phen ( see figure 2 ) . the conjugates were prepared by mixing 18 pmol 
 of qd550 or qd580 ( unless otherwise indicated ) with the desired equivalents 
 of ru - phen - labeled peptide in 100 l of pbs and 
 allowing the mixture to stand in a 1.5 ml microcentrifuge tube at 
 room temperature for 1 h prior to aliquoting into a microtiter well 
 plate ( or cuvette ) for spectroscopic measurement . ( a ) comparative absorption 
 spectra for qd550 , qd580 , and p1(ru - phen ) 
 measured at the concentrations indicated . ( a ) excitation wavelength 
 ( 400 nm ) for pl intensity measurements and ( b ) pump wavelength ( 420 
 nm ) for fsta measurements . representative tem micrographs of ( b ) qd550 
 and ( c ) qd580 samples . plots of quenching efficiency , e , versus the average number of ru - phen 
 peptides per qd , n , were fit with eq 1 using profit ver . 6.2.7 ( quantum soft , uetikon am see , switzerland ) 
 to determine the quenching rate constant , kq , where i is the actual number of 
 ru - phen - labeled peptides per qd and 0 the unquenched pl lifetime of the qds . qd pl decays 
 were fit with a biexponential function , eq 2 , and the amplitude - weighted lifetime calculated using eq 3.23 fsta 
 spectroscopy measurements of qd550 and qd580 were done using photoexcitation 
 at 420 nm in a manner similar to that described previously . qds ( 0.52.0 m ) were mixed with 40 equivalents of 
 p1(ru ) in pbs . control samples included solutions with 
 only qd550 or only qd580 ( 0.52.0 m ) and solutions with 
 only p1(ru ) ( 500 m ) . experiments with lower concentrations 
 of p1(ru ) had poor signal - to - noise ratios , particularly 
 in the red region of the spectrum . samples for fsta were prepared 
 fresh each day , and the data are an average of three replicates . some 
 analysis was done using single value decomposition ( svd ) with global 
 fitting using ultrafast systems llc software . the fsta instruments 
 were based on a commercial amplified ti : sapphire laser system ( 1.7 
 khz ; spectra - physics mira oscillator and spitfire pro amplifier ) housed 
 at the center for nanoscale materials at argonne national laboratory 
 or at the center for functional nanomaterials at brookhaven national 
 laboratory . a small amount of the amplifier output was used to generate 
 the white light continuum probe , and the remaining 95% went through 
 an optical parametric amplifier ( opa ) to produce 420 nm excitation 
 pulses at 0.7 j / pulse . the probe was delayed relative to 
 the pump on a mechanical delay line , and the pump beam was chopped 
 at half the repetition rate of the laser so that the absorption change 
 ( a ) could be measured as a function of delay 
 time , where a = log(ipump+probe / iprobe ) . the data 
 were chirp - corrected using a solvent blank to within 100 fs over the 
 spectral acquisition range ( 440770 nm ) . the samples were placed 
 in a 2 mm quartz cuvette and stirred during the acquisition . 120 fs . the transient absorption 
 changes at a particular probe wavelength , as a function of time , were 
 analyzed by fitting the kinetics with a multiexponential model convoluted 
 with a gaussian instrument response function having a 200 fs full - width - at - half - maximum 
 ( fwhm ) . qd pl quenching 
 on the subnanosecond - to - nanosecond time scale was also confirmed using 
 time - resolved pl upconversion ( upl ) experiments at the ultrafast laser 
 user facility at the center for functional nanomaterials at brookhaven 
 national laboratory . conditions for analyzing the qd580 with ru include 
 a 420 nm pump , 0.6 j / pulse in 1x pbs with emission detected 
 at 565 nm . model peptides with the same sequences as pron(ru ) and aibn(ru ) were constructed using the structure building 
 tools in 
 ucsf chimera version 1.4.1 . for the polyproline 
 peptides ( n = 0 , 3 , 6 , 12 , or 18 ) , the peptide models 
 were constructed based on previous work . for the poly(2-aminoisobutyric acid ) ( aib ) peptides ( n = 0 , 3 , 6 , or 9 ) , a similar approach was taken starting with the 
 coordinates of available aibn structures 
 in the protein data bank ( www.rcsb.org ) . energy minimization 
 was carried out in chimera using built - in features including antechamber 
 version 1.27 and the am1-bcc method of calculating charges . the polyproline helix was folded into a type 
 ii helix with / angles of 75/150. the aibn helix was 
 folded into a 310 helix with / angles of 
 49/26. some representative structures 
 derived during this modeling process are shown in the supporting information ( si ) . figure 2 shows the ground state absorption spectra of qd550 and qd580 , 
 which have first exciton peaks ( corresponding to band edge transitions ) 
 at 530 and 562 nm , respectively , and p1(ru ) , which has 
 an absorption maximum at 490 nm . the ru - phen 
 complex has only a very small absorption cross - section as compared 
 to the qds , and the significant differences in concentration between 
 ru - phen and qd species utilized in figure 2a amply illustrate this difference ( i.e. , the ru - phen is 140 times more concentrated than qd in that plot ) . 
 on average , the qd550 has a 4.5  0.7 nm diameter ( 2.5 nm diameter 
 cdse core and a 1.0 nm thick zns shell ) , while the qd580 has a 4.8 
  0.8 nm diameter ( 3.3 nm diameter cdse core and a 0.7 nm thick 
 zns shell ) , as extrapolated from the synthetic conditions and tem 
 micrographs ( average of > 100 qds with standard deviations of 15% ; 
 see figure 2 ) . figure 3 shows pl emission spectra for the qd550 and qd580 , highlighting 
 a progressive quenching response as the number of assembled p1(ru ) per qd increases . p1(ru ) conjugates 
 were excited at 400 nm , which minimized optical excitation of the 
 ru - phen . interestingly , these two qd materials exhibited 
 different trends in their quenching , with the qd580 being quenched 
 more strongly than the qd550 . the data in figure 3 were fit to eq 1 to determine the relative 
 quenching rates , kq0 . measurement of the qd pl lifetimes then permitted extraction 
 of the quenching rate constants , kq . the qd550 and qd580 pl decays ( data not shown ) were fit with 
 a biexponential decay , and the amplitude - weighted average lifetime 
 was taken as 0 . the qd pl decay constants and the 
 quenching rate constant for p1(ru ) are listed in table 2 for each of the native qd samples . the p1(ru ) quenches the qd580 ca . 20-fold more efficiently than qd550 , 
 with a rate comparable to the intrinsic excited state decay rate of 
 the qd580 ( 1.1  10 s ) . although 
 much slower charge transfer quenching was observed with the qd550 , 
 its intrinsic decay rate ( 8.6  10 s ) was similar to that of the qd580 , and the degree of this quenching 
 can be seen in figure 3b . the latter value 
 is suggested to be dominated by the qd s much slower and less 
 prominent lifetime component in a complex manner . quenching of ( a ) qd580 
 and ( b ) qd550 pl as a function of the assembly 
 of increasing amounts of p1(ru ) per qd , photoexcited 
 at 400 nm . ( c ) quenching efficiency as a function of the average number 
 of p1(ru ) per qd . the slightly negative quenching efficiencies 
 at low numbers of p1(ru ) per qd550 are due to a small 
 increase in the qd quantum yield as peptide is assembled before quenching 
 by the ru - phen becomes significant . this phenomenon is 
 often seen with the assembly of proteins onto the qd surface and is 
 believed to arise from surface passivation effects . similar to our initial findings , analysis 
 of the pl spectra revealed that both qd550 and qd580 exhibited pl 
 quenching that was independent of the emission wavelength ( see si , figure s1 ) . the qd580 ( full width at half - maximum , 
 fwhm = 32 nm ) exhibited only a < 2% relative standard deviation 
 ( rsd ) in wavelength - dependent quenching efficiency within 30 
 nm of the peak pl . the qd550 ( fwhm = 27 nm ) exhibited a slightly larger 
 rsd of ca . 10% ; however , this can be attributed to the effect of instrumental 
 noise in conjunction with the lower overall quenching efficiencies 
 compared to the qd580 . importantly , the slopes of the lines of best 
 fit for the wavelength - dependent quenching efficiencies were insignificant : 
 0.03  0.01% and 0.03  0.04% for the qd580 and qd550 , respectively . wavelength - dependent qd quenching would provide evidence of underlying 
 spectral overlap with a proximal acceptor , and this , in turn , would 
 be indicative of fret - based energy transfer processes . this would help 
 confirm whether the ru center was acting as an electron donor or acceptor 
 by chemically changing its ability to perform this function . this , 
 in turn , could also provide evidence for the directionality of charge 
 transfer . conversion of the p1(ru ) to p1(ru ) via oxidation with ce(aq ) was confirmed by loss of 
 the visible absorption band associated with ru - phen , 
 as shown in figure 4a . upon assembly of p1(ru ) with qd580 , little or no quenching of pl was observed ( figure 4b , c ) . a parallel control experiment with the assembly 
 of equivalent amounts of ru - phen again showed strong 
 quenching ( kq = 4.9  
 10 s ; the small discrepancy with the 
 value in table 2 is attributed to a different 
 batch of dhla - peg coating , as discussed later ) . ( a ) ground state absorption 
 spectra of ru - phen and 
 ru - phen ( obtained from the oxidation of the ru - phen with 
 ce ) . ( b ) quenching of qd580 pl with and without 10 
 equiv of ru - phen or ru - phen . ( c ) quenching 
 efficiency as a function of the number of ru - phen- or 
 ru - phen - labeled peptides assembled per qd580 . the latter 
 does not effectively quench the qd pl . in a final set of steady - state quenching experiments , we investigated 
 the effect of peptide length on the charge transfer quenching rate 
 constant . polyproline ( pron ) and poly(2-aminoisobutyric 
 acid ) ( aibn ) peptides were used for this 
 purpose and assembled with qd580 at average ratios varying from 0 
 to 18 . the quenching rate 
 peaked at n = 3 for both the pron(ru ) and aibn(ru ) and generally decreased for higher values of n. however , the quenching rate did not exhibit an exponential decrease 
 as the peptide length increased , as might be expected a priori . qd surface to ru center estimated 
 from structural modeling as described in the experimental 
 section . the data 
 for aib9 are 
 poorly fit by eq 1 , thus approximate minimum , 
 maximum , and average distances for the qd  ru - phen 
 separation were also considered . transient absorption 
 ( ta ) measurements of qd550 , qd580 , and their p1(ru ) conjugates 
 are summarized in table 4 and figure 5 . the ta spectra of the qd550 and qd580 showed similar 
 ground state recovery kinetics with nearly 3040% of the initial 
 excited state population decaying biexponentially within 100 ps . the 
 remaining 6070% of the decay was a long - lived component with 
 an average lifetime of 6 ns , which approached the longest lifetime 
 that could be measured by the ta systems . interestingly , the amplitude - weighted 
 excited state lifetimes for the qd550 and qd580 were indistinguishable , 
 and the differences between the individual decay components were within 
 the uncertainty of the experiment . with cdse qds , who suggested that this feature 
 could be indicative of dark excited states . with the addition of an average of 40 p1(ru ) per qd , 
 the ta signature for the qd ground state recovery changed moderately , 
 with a decrease in the amplitude - weighted excited state lifetime to 
 ca . forty equivalents 
 of p1(ru ) were used to ensure saturation of the charge 
 transfer quenching effect with both qds . no changes in the ru - phen ta spectrum could be unambiguously detected because 
 the ru - phen was very weakly absorbing , even when used 
 at relatively high ratios / concentrations . ( a ) ta spectra of 40:1 
 ru - phen - pep : qd550 in pbs buffer , photoexcited 
 at 420 nm . the inset on the right is the ground state recovery trace monitored 
 at 489 nm . ( b ) ta spectra of 40:1 ru - phen - pep : qd580 nm in pbs buffer , 
 photoexcited at 420 nm . the inset on the right is the ground state recovery 
 trace monitored at 562 nm . kinetic fits are a sum of exponentials 
 with a gaussian 250 fs instrument response function and based on the 
 standard use of a(t ) = irf 
 + ( i3aie ) . data are an average of five kinetic analyses 
 over the range from 520 to 560 nm , and the percentage of each amplitude 
 component is indicated by an ( % ) . a key feature of this study was having the ru - phen moiety 
 coupled to a peptide linker which , in turn , could be reliably and 
 controllably self - assembled to the qds . the p1 peptide sequence , csgaaaglshhhhhh , 
 is shown as divided into three functional modules : the n - terminal 
 cysteine residue provided a unique thiol group for labeling with ru - phen 
 maleimide and acts as the linker between these disparate entities ; 
 the intermediate sgaaagls sequence functioned as a spacer that was 
 able to pivot on the flexible gls portion ; and the c - terminal hexahistidine ( his6 ) assembled and anchored 
 the peptide to the qd ( figure 1 ) . his6-appended peptides , proteins , and even oligonucleotides can self - assemble , 
 via spontaneous metal - affinity coordination , to the zns surface of 
 cdse / zns qds functionalized with a variety of coatings . the binding interaction is characterized by rapid 
 assembly ( seconds to minutes ) , a high affinity equilibrium binding 
 constant ( ka  1  10 m ) , control over the number of biomolecules 
 assembled per qd ( i.e. , conjugate valence ) on the basis of stoichiometry , 
 and at least some control over their orientation . here , the his6 sequence binds to the qd 
 surface , while the gls can adopt a conformation that extends the rest 
 of the peptide sequence away from the qd surface and into the surrounding 
 layer of peg ligands . the his6 also provided another critical 
 function in that it allowed for facile purification of labeled peptide 
 using immobilized metal - affinity chromatography ( imac ) . the other peptides used in this study were the 
 helix - forming pron and aibn sequences , which offer some ability 
 to incrementally control the distance between the qd and ru - phen . this arises as the helices for both motifs are predicted to 
 be quite rigid and should minimize bending of the peptide toward the 
 qd surface . furthermore , the use of a bulky pegylated surface ligand 
 will also contribute in a steric manner toward directing the peptides 
 away from the qd surface . it is important to note , however , that this still does 
 not completely preclude some peptides assuming bent or otherwise altered 
 conformations which could affect charge transfer . as shown in figure 1a , the conjugation of the ru - phen to 
 the qds using the his6-terminated peptide is expected to 
 result in a centrosymmetric arrangement or display of the ru - phen 
 around the qd ( albeit that the peptide should have some conformational 
 freedom ) . comparison of this peptide 
 to similar sequences used in a prior fret study suggests that the 
 p1 peptide assembly places the ru - phen ca . the distance 
 is within the linear extension of the dhla - peg ligands on the surface 
 the qd . previous work has also shown that qds of similar size , and 
 functionalized with dhla - based ligands , can display a maximum of ca . , we use an average of 40 peptides per qd , 
 which , although slightly below saturation , is still sufficient to 
 elicit a significant qd quenching response . it is also important 
 to note that the ru atom interaction with the phenanthroline moiety 
 is considered a coordination metal complex with coordinate covalent 
 bonds . the coordinate covalent bond is a chemical bond in which both 
 bonding electrons are from the ligand , and such phenanthroline complexes 
 are considered very stable . our initial 
 work also demonstrated that the free ru - phen complex has a very small 
 direct interaction with the pegylated qds utilized here due to electrostatic 
 interactions , and this interaction could 
 be counteracted by the presence of nacl in pbs as done here . from 
 a practical standpoint this means that , at the experimental concentrations 
 utilized here , the ru - phen complex does not significantly contribute 
 to qd quenching via direct physical interactions with the qd surface , 
 nor will the ru atom dissociate from the phenathroline complex which 
 cumulatively alleviated our concerns about these issues . we have previously measured the cyclic voltammograms 
 of cdse / zns qds coated with dhla - peg ligands and found a broad , irreversible 
 oxidation peak at eox0 = 0.22  0.2 v ( vs ag / agcl ) . a prior estimate also places the conduction 
 band and valence band edges of the qds between 1.1 to 1.2 
 v and 0.8 to 0.9 v ( vs ag / agcl ) , respectively . a cyclic voltammogram for the ru - phen is shown 
 in the si ( figure s5 ) and exhibited two 
 broad irreversible reduction peaks with cathodic peak potentials ( epc ) centered at 1.16 and 1.33 
 v ( vs ag / agcl ) , as well as the reversible ru / ruredox couple at e1/2 = 314 mv ( vs ag / agcl ) . this half - wave potential is intermediate to the expected conduction 
 and valence band edges of the qds and slightly higher in energy than 
 the oxidation level(s ) of the qds . this alignment is , a priori , favorable 
 for hole transfer from the qd to the ru - phen . in our 
 previous study , the peptide - linked assembly of metal complexes with 
 oxidation potentials intermediate to the expected band edges , but 
 also lower in energy than the oxidation levels of the qd ( e.g. , ferrocene ) , 
 did not cause quenching of qd pl . these 
 oxidation states , which are thought to correspond to surface states , 
 are critical for charge transfer quenching . the quenching 
 of qd pl via ru - phen - labeled peptides has been previously 
 attributed to electron transfer from the ru to the qd 
 with the suggestion of concomitant hole transfer from the qd . this mechanism of charge transfer was inferred 
 from ground state absorption spectra and consideration of the redox 
 levels for the qd and ru - phen . in general , charge transfer 
 quenching efficiency was observed to increase with decreasing qd size 
 and with a greater average number of ru - phen per qd . not surprisingly , surface ligand character also appeared to significantly 
 influence the quenching behavior as well . here , we again observed that quenching efficiency increased as the 
 number of proximal ru - phen displayed around the qd increased . however , we also observed that two qds of similar size ( 4.5  
 0.7 nm diameter for core / shell qd550 and 4.8  0.8 nm for qd580 ) 
 and modified with the same surface ligands can manifest vastly different 
 quenching efficiencies : cdse / zns qd580 had a quenching rate constant 
 of kq  8  
 10 s , which was an order of magnitude 
 larger than the kq  
 4  10 s associated with cdse / zns 
 qd550 and the same peptide , p1(ru ) . the qds had comparable 
 pl lifetimes , measured as 9 ns ( qd580 ) and 12 ns ( qd550 ) , 
 identical quantum yields ( 14% ) , and neither exhibited any significant 
 defect emission . the discrepancy between the qd550 and qd580 quenching 
 efficiencies , despite their similar macroscopic pl properties , suggests 
 that the structure of the qd interface is critically important in 
 determining charge - transfer - based quenching efficiency . one such difference 
 here was that the qd580 samples had a shell 0.3 nm thinner than the 
 qd550 . given that the shell is expected to be one of the principal 
 impediments to charge transfer between the ru - phen and 
 qd core , it appears that angstrom - scale changes in shell thickness , 
 or more uniform coverage that appears as a thicker shell , can cause 
 significant changes in the rate of charge transfer in these samples . 
 in support of this hypothesis , zhu et al . have reported that the rate 
 of electron transfer from cdse / zns qds to an adsorbed dye , in a hydrophobic 
 system , has an exponential dependence on the shell thickness . in contrast to the putatively strong influence 
 of zns shell thickness , the distance dependence of the quenching effect 
 arising from just peptide extension alone appears to be rather modest , 
 despite charge transfer quenching occurring over qd surface - to - ru - phen distances that are estimated to vary between ca . the aibn series of peptides 
 are chemically different from pro and are expected to allow the ru - phen to maintain a slightly closer approach to the qd surface . 
 however , it is important to note that through - bond charge transfer 
 from metal centers across peptide structures can be quite complex 
 with the possibility of different residues providing either neutral , 
 conductive , or insulative properties . the lack of significant quenching effects versus distance with these 
 peptides may be more reflective of their inherent properties and environment 
 or , alternatively , may reflect the more dominant role of shell size 
 effects on charge transfer . although certainly worthy of future study , 
 the effect of the peptide sequences / residues on charge transfer processes 
 in these systems is beyond the current scope . in our initial study , steady - state 
 and time - resolved fluorescence 
 on the nanosecond time scale was used to extensively study the quenching 
 of qd pl with several metal complex peptide conjugates that 
 varied in oxidation potential including the current ru - phen  peptides . when qds were assembled with redox active molecules 
 that did not have favorable energy matching for charge transfer , only 
 a minor amount of pl signal was quenched ( < 20% ) with no change 
 in the qd lifetime . critically , it was shown that qd pl could only 
 be efficiently quenched by the ru - phen  peptide , the only complex 
 with a favorable energy level match to the qd donor . the results 
 in figure 4c , which show that ru - phen does not elicit any significant quenching effect when brought 
 into proximity of the qd , unambiguously demonstrate that a ru center is required to quench qd pl . the homo electron of 
 ground state ru - phen is thus integral to the quenching 
 mechanism . this result is consistent with the previously posited mechanism 
 for hole transfer from the qd 1sh state to the ru - phen ( i.e. , electron transfer from the ru - phen homo ) . however , as shown in figure 4a , the oxidative 
 transition between ru - phen and ru - phen also 
 changes the absorption spectrum of the complex , potentially resulting 
 in a change in the spectral overlap integral relevant to a fret mechanism 
 of quenching ; the latter process has recently been reported for similar 
 qd  peptide bioconjugates labeled with os complexes 
 and c60 . in the current example , the overlap decreases from 2.2  10 and 1.4  10 cm mol for ru - phen with qd550 and 
 qd580 , respectively , to < 0.7  10 cm mol for ru - phen with either 
 qd . these overlaps for ru - phen are between 10- and 100-fold 
 smaller than those observed for typical qd  dye fret pairs . to obtain a fret efficiency of 75% with 
 4 p1(ru ) per qd , the ru - phen would need 
 to be located 3.0 nm from the center of the qd , which is not 
 physically possible with the qd580 ( core radius 3.3 nm from 
 tem , plus a 0.7 nm thick shell ) . further , the quenching rate for p1(ru ) with the qd550 should be 20% larger than with qd580 ; however , 
 this was not observed , and neither qd exhibited wavelength - dependent 
 quenching efficiency when conjugated to and quenched by the ru - phen , as would be expected for fret . indeed , wavelength - dependent quenching can be considered a hallmark 
 of qd quenching by fret . a preliminary conclusion 
 to the same effect was also reached in our previous examination of 
 qd  ru - phen interactions . given the foregoing , a fret mechanism of quenching can be conclusively 
 ruled out . cumulatively , quenching appears to be through electronic 
 interactions between ru - phen  peptide and the linked qd , and 
 in conjunction with the above , hole transfer from the qd to the ru - phen therefore becomes a more probable mechanism of quenching . the excited state 
 dynamics of cdse and cds qds have been previously examined under various 
 conditions and solvents using ta spectroscopy . direct photoexcitation of the qd results in a ground state bleach 
 of the 1s state and the corresponding intraband electronic transition 
 in the visible region . the time scale of this process is on the order 
 of hundreds of femtoseconds with a multiexponential recovery that 
 is attributed to conduction band filling and surface trapping of charges 
 that persist on the nanosecond to microsecond time scale . for example , electron transfer from qds to an 
 adsorbed viologen dye was identified through near complete bleaching 
 of the qd s band - edge ta signal and parallel observation of 
 a spectral signature for the reduced form of the adsorbed dye . hole transfer from qds to adsorbates has been 
 more difficult to identify from ta spectra , relying on spectral signatures 
 from the oxidized adsorbate , because the qd ta spectra 
 reflect conduction band electron dynamics that are not involved in 
 the initial transfer . however , a state - resolved approach has been 
 described by kambhampati and co - workers that resolves dynamics through 
 selective state pumping and probing . when 
 qds are photoexcited directly into specific states , the electron relaxation 
 dynamics , hot exciton relaxation dynamics , and transitions from first 
 excited states for holes and electrons can be resolved . more recently , 
 time - resolved pl experiments in conjunction with ta have provided 
 insight into hole dynamics indirectly . for example , sykora et al . 
 reported that the pl of hydrophobic cdse qds coated with trioctylphosphine 
 oxide and adsorbed ru polypyridine complexes are quenched through 
 hole transfer from the qd to the ru  polypyridine complex . this conclusion was arrived at through both 
 fsta studies of the charge carrier dynamics and time - resolved pl upconversion 
 ( upl ) experiments . the carrier dynamics were characterized by a 5 
 ps depopulation of the qd hole states ; however , the distance between the qd and adsorbed ru  polypyridine 
 complex was extremely small and was not varied . here , the confounding 
 issue is that the ta spectra showed no features that could be unambiguously 
 attributable to the oxidized ( ru ) or reduced ( ru ) ru - phen complex . despite the strong structural similarities 
 to ru complexes with 2,2-bipyridine ligands , 
 the spectra of 1,10-phenanthroline ( phen ) excited * mlct 
 states as bis- and tris - phen ligands the ta spectrum of the ru - phen - labeled peptide alone , 
 shown in si figure s3 , exhibits a positive 
 transient feature between 550 and 700 nm . this absorption has a biexponential 
 decay ( 1 = 16 ps and 2 = 700 ps ) , 
 where the 700 ps decay matches the monoexponential recovery of the 
 ground state bleach at 472 nm . comparing the ta spectra between the 
 qd580 alone and its assembly with ru - phen , small spectral 
 differences in the ground state bleach were observed , which were then 
 analyzed by single value decomposition ( svd ) with global fitting to 
 reveal a 5 ps component that corresponded to the ru - phen 
 ground state bleach at 472 nm ( see figure s4 , si ) . it is likely that direct photoexcitation of ru - phen at 420 nm leads to a minor population of * ru - phen . in experiments with qds , the ru - phen - labeled peptide 
 concentrations were below 100 m , and at this concentration 
 they would have contributed < 5% of the total signal at 530 nm , 
 a value that is essentially negligible . the ta spectrum of the qd580-p1(ru ) assemblies showed a positive absorption feature ( a  10 ) between 550 and 700 nm . 
 the ta spectrum for the qd550-p1(ru ) , however , shows 
 no such feature within the experimental precision . notwithstanding 
 the lack of evidence for fret in other experiments , the positive ta 
 feature is not sufficiently pronounced to be consistent with fret , 
 in contrast to recent results from a previous assembly consisting 
 of qds paired with os  peptidyl complexes . within the qd550ru - phen assemblies , 
 charge transfer occurred 
 after photoexcitation with a recovery half - life of 1/2 = 1.3 ns in contrast to the 1/2 = 383 ps for the 
 qd580ru - phen assemblies . interestingly , the qd580 pl was quenched 
 more strongly than the qd550 pl and exhibited a faster recovery . the 
 qd bleach kinetics , however , only provide insight into the dynamics 
 of conduction band state electrons and do not reflect hole dynamics . retention of the qd bleach suggests no substantial 
 conduction band electron dynamics occur on the ultrafast time scale . previously , huang et al . found that cdse qds with adsorbed re(co)3cl(dcbpy ) ( dcbpy = 4,4-dicarboxy-2,2-bipyridine ) 
 ( ered0 = 1.0 vs ag / agcl ) undergo photoinduced electron transfer 
 from the conduction band of the cdse qds to the re complex with 1/2 = 2.31000 ps depending on core size . this analysis was based on fitting the qd ground 
 state bleach kinetics . since the electronic absorption of the ru - phen was not observed in ta spectra , the lifetimes of electron 
 transfer and ground state recovery were estimated from the kinetics 
 of the spectral bleaching traces recorded between 520 and 560 nm for 
 the ru - phen assembled to the qd550 and qd580 . this region 
 corresponded to the maximum qd bleach , with minimal contribution from 
 the ru - phen ground state . again , we note that extensive 
 time - resolved fluorescence data were 
 collected in our initial report on the nanosecond time scale when 
 a significant population of the excited state has been quenched . this explains why a smaller population of quenching 
 of the qd exciton state is observed by the femtosecond transient absorption 
 experiments here . qd pl quenching on the subnanosecond - to - nanosecond 
 time scale was also confirmed using time - resolved pl upconversion 
 ( upl ) experiments . figure 6 shows a rather 
 slow quenching process , and the rate of this quenching lies on a time 
 scale that is not accurately measured because of the ta and upl delay 
 tracks . using figure 6 , the results observed here 
 are consistent with the slower dynamics expected when the distance 
 of the qd and ru - phen  peptide is lengthened . in contrast , the 
 aforementioned studies by sykora measured the charge transfer dynamics 
 using upl and fsta on qds with electrostatically adsorbed ru in summary , we build from our previous reports demonstrating the 
 quenching of qd pl by proximal , peptide - linked , ru - phen 
 complexes . although a charge - transfer process 
 was implicated , we were unable to unambiguously define the mechanism 
 or stipulate the directionality of electron and/or hole transfer . 
 here , we contribute more to the understanding of qd quenching by this 
 charge transfer process . first , we find that resonance energy transfer 
 can be conclusively ruled out as the mechanism of quenching . comparison 
 of qd interactions with ru - phen and ru - phen - labeled 
 peptides further reveals that the ru state is essential 
 for qd quenching . this result provides strong indirect evidence for 
 hole transfer from the qd to the ru - phen , which , importantly , 
 is mechanistically feasible and consistent with all experiments to 
 date . the difference in pl quenching efficiency between qd550 
 and qd580 , 
 along with a comparison among ru - peptides of differing 
 persistence length , suggests that shell thickness is a most critical 
 determinant of charge transfer rates in these assemblies . this observation , 
 in conjunction with our earlier observations that qd surface coating 
 ( i.e. , negatively charged dhla versus neutral dhla - peg ) contributed 
 to observable differences in ru - phen charge - transfer - based 
 processes , suggests that the interfacial 
 environment of the qd strongly influences charge transfer quenching 
 efficiency . this result is not unexpected given the growing literature 
 describing such phenomena . the qd interface , especially in an aqueous buffer , is a complex 
 environment with the possibility of both major and subtle contributions 
 from crystal facets , shell coverage , ligand densities , and solvation , 
 along with other complicating factors such as localized ph gradients 
 at the qd surface . fsta and upl analysis reveals that the rate of 
 qd quenching is slow on the ultrafast time scale and occurs in the 
 nanosecond rather than picosecond regime . this analysis is , however , 
 complicated by the lack of an unambiguous spectroscopic signal from 
 the ru - phen . interestingly , these findings are mechanistically 
 similar to those reported by huang et al . for cdse qds interacting 
 with an adsorbed re compound . this is 
 also consistent with the rate of hole transfer in other qd assemblies 
 including those described in refs ( 25 and 71 ) . we do note that our system is more complicated than many others 
 because it was constituted in the high - salt , aqueous environment typically 
 needed for biosensing . further experiments will focus on pairing differently 
 functionalized qds with ru - phen and other redox - active 
 metal complexes in a similar environment to try to elucidate the contributions 
 of the solubilizing ligands and localized environment . a mechanistic 
 picture of the charge transfer quenching of biologically 
 relevant , core / shell qd bioconjugates with ru - phen may 
 provide a means of creating qd probes that have improved sensitivity 
 and greater versatility for detecting a more diverse range of biological 
 processes and analytes or , alternatively , that respond to biochemical 
 redox stimuli rather than biomolecular interactions . determining the direction of charge transfer and identifying 
 the states involved remains critically important to this endeavor 
 as it may enable the design of systems where charge transfer is  intercepted  
 by species in biologically relevant redox pathways , resulting in light - up 
 sensors that transduce , for example , cellular metabolism . the detection 
 and visualization of redox pathways in biological systems still remains 
 exceedingly challenging . as noted , qds are already 
 well developed for fluorescent cellular and small animal labeling and for fret - based sensing . when better understood , charge transfer interactions 
 with qds may add a new dimension to such sensing and imaging applications 
 and open up new avenues for probing biological systems .
charge transfer processes with semiconductor 
 quantum dots ( qds ) 
 have generated much interest for potential utility in energy conversion . 
 such configurations are generally nonbiological ; however , recent studies 
 have shown that a redox - active ruthenium(ii)phenanthroline 
 complex ( ru2 + -phen ) is particularly efficient at quenching 
 the photoluminescence ( pl ) of qds , and this mechanism demonstrates 
 good potential for application as a generalized biosensing detection 
 modality since it is aqueous compatible . 
 multiple possibilities for 
 charge transfer and/or energy transfer mechanisms exist within this 
 type of assembly , and there is currently a limited understanding of 
 the underlying photophysical processes in such biocomposite systems 
 where nanomaterials are directly interfaced with biomolecules such 
 as proteins . here , we utilize 
 redox reactions , steady - state absorption , pl spectroscopy , time - resolved 
 pl spectroscopy , and femtosecond transient absorption spectroscopy 
 ( fsta ) to investigate pl quenching in biological assemblies of cdse / zns 
 qds formed with peptide - linked ru2 + -phen . 
 the results reveal 
 that qd quenching requires the ru2 + oxidation state and 
 is not consistent with frster resonance energy transfer , strongly 
 supporting a charge transfer mechanism . 
 further , two colors of cdse / zns 
 core / shell qds with similar macroscopic optical properties were found 
 to have very different rates of charge transfer quenching , by ru2 + -phen with the key difference between them appearing to be 
 the thickness of their zns outer shell . 
 the effect of shell thickness 
 was found to be larger than the effect of increasing distance between 
 the qd and ru2 + -phen when using peptides of increasing 
 persistence length . 
 fsta and time - resolved upconversion pl results 
 further show that exciton quenching is a rather slow process consistent 
 with other qd conjugate materials that undergo hole transfer . 
 an improved 
 understanding of the qd 
 ru2 + -phen system can allow 
 for the design of more sophisticated charge - transfer - based biosensors 
 using qd platforms .