there are two basic 
 strategies for constructing devices in which 
 the flow of electrons is mitigated by single molecules : top - down and 
 bottom - up . top - down methods rely on nanofabrication or mechanical 
 control to form the nanometer - scale junctions between  top  
 and  bottom  electrodes necessary to contact molecules 
 end - to - end . bottom - up methods typically rely on the self - assembly 
 of molecules onto a bottom electrode in which the junction is defined 
 by the molecules themselves when a top electrode ( top - contact ) is 
 applied ; these are devices that , in part , fabricate themselves . nature provides a plethora of intricate molecular 
 complexes that form by and are prone to self - assembly ; however , these 
 complexes are subject to different constraints than their simpler , 
 synthetic counterparts such as the well - studied alkanethiols . conventional techniques for forming top - contacts 
 for devices or for measuring electrical properties over large areas 
 of self - assembled monolayers ( sams ) of organic molecules have been 
 thus far limited to the direct deposition of metals by electron - beam or thermal evaporation or the addition 
 of an electrically conductive polymer layer between the sam and a 
 metallic top contact ; however , in these techniques , 
 the high temperatures , vacuum processing and the need for acidic or 
 organic solvents to spin coat the polymers are not ideal for biomacromolecules . other ,  soft ,  nondamaging methods for forming top - contacts , 
 such as the use of hanging hg drops ( hmds ) , 
 are better suited to this task . ron et al . used hmds as a nondamaging 
 method for forming top - contacts to measure the electron transfer ( et ) 
 through three protein ( complexes ) on doped - si substrates : azurin ( az ) , 
 bacteriorhodopsin ( br ) , and bovine serum albumin ( bsa ) , establishing 
 that proteins can function as building blocks for bottom - up tunneling 
 junctions . although nondamaging , hg is better suited for si substrates 
 than metals , with which it tends to form amalgams ( au and ag are the 
 most common metals used in bottom - up tunneling junctions ) . these studies 
 on complete proteins were preceded by discussed models of mechanisms 
 of electron transport through peptides and proteins , for example , 
 by giese et al . waleed shinawari et al . reviewed biomolecular charge transport , with 
 a particular focus on dna and protein molecules . a recent progress 
 report by amdursky et al . compares current 
 densities for different techniques , including cp - afm and scanning 
 tunneling microscopy ( stm ) and macroscopic , for junctions comprising 
 proteins and small molecules . the liquid metal eutectic , ga  in 
 ( which we abbreviate  egain  ; 
 75% ga , 25% in by weight , mp = 15.5 c ) , can function similarly to hg in the hmd method , but does not form 
 alloys with noble metals . it also exhibits 
 non - newtonian rheology , driven by a self - limiting oxide , and does not spontaneously reorganize to minimize 
 its surface energy . thus , it can be formed 
 into stable , nonspherical tips to form 100 m contacts and is stable in microfluidic channels , which facilitates 
 variable - temperature measurements . photosystem i ( psi ) is a complex that houses 
 one of the two reaction 
 centers used in the photosynthetic reactions in cyanobacteria , algae 
 and plants where the conversion of solar energy to electron hole pairs 
 takes place . isolated psi complexes from thermophilic unicellular 
 cyanobacterium thermosynechococcus elongatus bp-1 have evolutionally formed a trimer structure for improved 
 light absorption efficiency and stability at harsh conditions . the 
 monomer of psi has a polar stroma and lumen and an apolar backbone . its size is approximately 13  8  9 nm and contains 96 
 light sensitive chlorophyll a ( chla ) molecules that are densely packed in the protein scaffold to harvest light . in the complex , harvested 
 photons are converted into excited electrons by chlorophylls at p700 
 ( via a special pair of chla molecules ) . this transfer 
 occurs from the primary electron donor complex to the primary electron 
 acceptor a0 ( chla ) , to the ferredoxine 
 docking region through the built - in electron transport chain a1 ( phylloquinone ) , fx , fa and fb ( fe4s4 clusters ) ( figure 1a ) . the complex has a photovoltage of 1 v under illumination 
 with an internal quantum efficiency close to unity.these features make psi a unique and interesting protein complex 
 to study . furthermore , the entire psi complex , as well as the reaction 
 center ( rci ) from plants and cyanobacteria have been extensively characterized 
 and the electrical properties have been observed by a variety of experimental 
 techniques . we studied the entire psi complex , not isolated rci , because it 
 contains the electron transport chain , the light - harvesting array 
 of chlorophylls and the protein scaffold that holds them in place . ( a ) schematic 
 representation of electron transfer chain in photosystem 
 i ( et chain ) . ( b ) possible orientations of psi on au surfaced induced 
 by chemical modification . orientation where the flow is in the opposite direction ( p700 adjacent 
 to substrate ) , a  down  orientation in which the natural 
 direction of the flow of electrons is toward the electrode surface 
 ( fb down ) , or with its electron transport vector parallel to the substrate . we investigated the j  v characteristics 
 of sams of psi in the dark on template - stripped au measured by forming top - contacts from tips of egain . egain 
 has been used to measure current density ( j ) versus 
 applied voltage ( v ) through sams of a variety of 
 molecules to form rectifiers , to observe quantum interference effects , to relate dipole moments to vacuum - level shifts , and it is sensitive enough to resolve the odd  even 
 effect in sams of alkanethiolates . unlike sams 
 of small molecules , however , there are no data from egain tunneling 
 junctions of psi or any other protein complexes against which to compare . 
 thus , we characterized sams of psi trimers by interrogating the complexes 
 in the dark , one at a time , by cp - afm and compared those data to the 
 ensemble averages produced by egain contacts in order to relate our 
 findings to previous studies . we use the asymmetry of the conductance 
 data ( i.e. , rectification ) to elucidate the orientation of psi because 
 it is self - referencing and , therefore , is less sensitive than the 
 magnitude of j to other structural features of the 
 sam , but very sensitive to the specific geometry of the sam / egain 
 interface ( e.g. , orientation ) . asymmetry 
 observed in current transport is particularly useful for elucidating 
 the transport properties of protein complexes , which are vastly more 
 complex in structure , size , self - assembly and electrical properties 
 than small molecules , because it eliminates the uncertainty of area 
 calculations that arises from topology , packing density , etc . and while virtually all tunneling junctions 
 with egain top contacts show some asymmetry ( because the junction 
 itself is asymmetric ) , the rectifying behavior of the sam is readily 
 distinguishable . we are not suggesting 
 that sams of psi make particularly good rectifiers of current the 
 magnitude of the asymmetry of the current is quite small  only 
 that small changes in asymmetry can provide information about the 
 orientation of psi complexes . orientation 
 in which the natural flow of photogenerated electrons is to the electrode 
 surface ( fb down ) , an  up  orientation where 
 electrons would flow in the opposite direction ( p700 adjacent to substrate ) , 
 or with its electron transport vector parallel to the substrate ( figure 1b ) . this level of control over the orientation of 
 the electron transport chain provides an opportunity to determine 
 its role in the tunneling transport through psi through the self - assembly 
 process rather than by modifying the complexes themselves . different 
 methods have been used to control the orientation of psi and rci on 
 surfaces , ranging from surface modification with different functional 
 groups that interact electrostatically with different parts of the 
 protein complex , to direct covalent attachment via mutation and sams with different functional head groups . with egain , we can address the monolayers of psi complexes electrically 
 and from these electrical measurements we can probe the average orientation 
 of the complexes in the monolayer to compare against afm images and 
 cp - afm i  v data . according to previous 
 reports on single complexes of rci ( i.e. , 
 not sams or ensembles and not psi ) , asymmetric charge transport is 
 completely dependent on the orientation on the surface of the electrode . they platinized one end of the photosynthetic complex 
 and  welded  it to a au surface using sams of small 
 molecules as  director  monolayers . they used scanning tunneling spectroscopy ( sts ) to examine 
 the electronic properties of each rci , which elicited orientation - dependent 
 asymmetry . this phenomenon is easily conflated with light - driven processes , which involves hopping transport ( to move through 
 the transport chain as the electron changes in energy ) and therefore 
 should not play a role in tunneling measurements . yet , the absolute 
 orientation of rci and , by extension , psi is assigned from sts data 
 based on the assumption that the direction of asymmetry ( rectification ) 
 follows the electron transport chain . by measuring 
 tunneling junctions comprising psi in the dark , we found that this 
 assignment of the orientation does not , in fact , map onto psi it 
 is backward  and that tunneling transport , therefore , likely 
 dominates any hopping contribution from the electron transport chain . we exposed ultraflat 
 template stripped 
 au ( au ) substrates to a solution of 1 mm 2-mercaptoethanol 
 ( 2me ) or sodium 3-mercapto-1-propanesulfonate ( mps ) to form director 
 sams  to bias the orientation of psi trimers that self - assemble 
 on top of these sams . these director sams differ in length by 1.7  , which may affect 
 the magnitude of the tunneling current , but we do not rely on this 
 magnitude as a measure in this work and small changes in the thickness 
 of the director sam are unlikely to influence asymmetry . the psi in 
 cyanobacteria has an asymmetric distribution of surface charges in 
 the stroma and lumen ( see supporting information for details ) . two thirds of all the charged surface residues are 
 concentrated at the  top  of the complex ; the stromal , 
 fab electron acceptor side ( figure 1a ) . this difference is likely what determines the preferred orientation 
 during self - assembly on modified surfaces . we immobilized psi on the 
 substrates by drop casting from aqueous buffer and incubating them 
 ( i.e. , leaving them in contact ) for 2 h. we investigated each sam 
 topographically by afm and electrically using cp - afm and egain . the 
 cp - afm data are averages of i  v curves from 
 100 complexes for each director sam . thus , they sample the distribution 
 of orientations similarly to the large - area egain measurements and 
 can be compared directly . we define the asymmetry of current transport 
 ( rectification ratio ) , r , as the ratio of j or i at positive to negative bias ; r = |j()/j(+)| 
 for egain and r = |i()/i(+)| for cp - afm , both with respect to the wiring convention 
 for cp - afm ( see supporting information ) . measured afm height profile images show better psi coverage for 
 2me than for mps , with protein heights close to 6 nm on au . this value is less than the 9 nm thickness derived from the crystal 
 structure data because these sams are measured under ambient , anhydrous 
 conditions and contact with the afm tip can compress them somewhat 
 ( for more details see supporting information ) . we imaged individual psi trimers within sams of psi formed on 
 both directing sams by afm to determine the density ( per m ) and measured their electrical properties by cp - afm to determine 
 the orientation of the complexes , assigning low values of r to psi that is oriented up and high values to down . values 
 of unity were assigned to psi that is oriented parallel to the substrate 
 ( sideways ) . ( the average values of r for egain and cp - afm 
 are given in table 2 . ) individual i  v traces were averaged over 100 psi complexes at different locations 
 within the sams . the results 
 were obtained by 
 measuring the asymmetry of the i  v curves 
 by cp - afm . the average coverage on an area of 1 m was calculated from afm height images . the table summarizes the 
 statistical distributions of the i  v curves 
 of over 100 points for both director sams . these values were calculated from 
 evaluating j  v curves at 
 1 v. r = |j()/j(+)| for egain and r = |i()/i(+)| for cp - afm , both with respect to 
 the wiring convention for cp - afm ( see supporting 
 information ) . values refer to the calculated geometric mean 
 from curves at 1 v. for egain measurements , the sams of psi on both director sams were 
 contacted by lowering a syringe ( connected to an electrometer ) supporting 
 a tip of egain . recent reports on egain junctions comprising alkanethiolates 
 employ a slightly different method in which the tips are first flattened 
 against a si wafer and then pressed into the sam ; however , light contact with as - prepared tips yielded stable 
 and reproducible results on sams of psi . we observed four different 
 behaviors : ( i ) shorts , characterized by linear j  v curves with currents in the a regime ; ( ii ) no - contact , characterized 
 by noisy currents in the pa regime ; ( iii ) poor - contact , which begins 
 with no - contact j  v curves , but is followed 
 by shorts after further lowering of the egain tip ; and ( iv ) good contact , 
 characterized by s - shaped j these data are summarized in table 3 and show higher rectification ratio when 2me is used as opposed 
 to mps , which we ascribe to the different distributions of orientations 
 of psi induced by the director sams . we analyzed the data identically 
 to the cp - afm data by averaging log j. see the supporting information for a detailed discussion 
 of the data analysis . the 
 values of j are the geometrical averages from measurements 
 with egain tips . the yield of working devices is based on devices with egain as top 
 electrode . as control experiments , we measured 
 the electrical properties of 
 denatured psi ( which are shown in the supporting 
 information ) . we boiled psi for 20 min at 99 c , which 
 is sufficient to denature it completely and then prepared sams of 
 deactivated psi by following the same procedures used to prepare sams 
 of active psi . we observed no rectifying behavior in egain junctions 
 comprising boiled psi , nor did we observe shorts ( table 3 ) . the lack of shorts implies that denatured psi still covers 
 the substrate , as sams of 2me and mps are fragile and give mostly 
 shorts . thus , the origin of the asymmetry is dependent on the presence 
 of intact psi structures ( i.e. , the overall asymmetry of the junction 
 depends on the presence of intact complexes of psi ) . we varied 
 the density ( surface coverage ) of the sams of psi by preparing psi 
 trimer solutions of different concentrations ( dilutions from 1:1 to 
 1:130 , ratio of concentration of 1 m psi to buffer ) while keeping 
 the incubation time fixed . from afm images , we determined the surface 
 coverage by comparing the psi - covered surface to the total surface 
 area ; psi= [ napsi / atotal]100% where n is the number density of psi , apsi is the area occupied by psi complexes ( 25 nm , determined from tem ) , atotal is the total area investigated . figure 2 shows the correlation between psi and the magnitude of j in egain junctions . the 
 exponential relationship between j and surface coverage 
 is further evidence that the charges are flowing through the psi complexes . 
 below 23% coverage the junctions become unstable , irreproducible , 
 and yield mostly shorts ( as do bare director sams ) . presumably this 
 percentage is the cutoff value below which egain is able to penetrate 
 between the psi complexes and contact the director sam . this behavior 
 is not unlike defect - mediated transport in which the electrical properties 
 of highly conductive defects ( space between psi complexes ) become 
 dominant at a critical density . the saturation 
 of j at 23% implies that transport occurs exclusively 
 through psi and that there is no ( or constant ) leakage current , which 
 allows the direct comparison of the data from egain and cp - afm junctions . asymptotic 
 fit of j at 1 v vs percentage of surface 
 coverage for psi on 2me as directing sam . the threshold limit of coverage is 23% as determined 
 by imaging surfaces at different psi : buffer concentrations and characterizing 
 their behavior with an egain tips . inset images show psi complexes 
 on au at different concentrations which correspond to 
 the percent coverages shown . the devices were imaged on an afm at 
 2.5 m . to evaluate the influence 
 of contact with tips of egain , we marked 
 areas of the sams of psi and imaged them by afm before and after forming 
 egain junctions and acquiring j we observed no qualitative 
 damage to the monolayers and , by counting the number of complexes 
 in each junction before and after forming egain junctions , determined 
 that no complexes were extricated from the monolayer . from these experiments we conclude that the sams of psi are not damaged and that the individual 
 complexes do not move and are not removed during measurement . thus , 
 egain is a demonstrably nondamaging method for investigating charge - transport 
 through sams of psi and is capable of forming reversible junctions , 
 which may be useful for characterizing sams of psi ( or other protein 
 complexes ) as an intermediate step in the fabrication of devices to , 
 for example , verify the orientation of the complexes . the rectifying behavior of rci 
 ( not psi ) was previously observed by sts and cp - afm on individual protein complexes . 
 in our studies with egain , psi assembled on director sams exhibited 
 an asymmetric conductance between bias voltages with r = 5.0 with 2me and 2.0 with mps ( table 2 ) . the direction of rectification does not invert when the orientation 
 of the complexes is reversed because there is some built - in asymmetry 
 in these junctions ; the bottom electrode supports a covalently bound 
 director sam that supports the psi complexes , while the top - contact 
 is physisorbed either via contact with an afm tip or by supporting 
 an egain electrode . thus , one orientation works with the built - in 
 asymmetry and the other against it , but not sufficiently to overcome 
 it completely . we ascribe the asymmetry in both cases to the 
 ratio of the orientation of the complexes ( table 1 ) . an important distinction between single - complex studies 
 ( e.g. , cp - afm and sts ) and large - area studies ( e.g. , egain and hmd ) 
 is that the latter sample the average orientation . coupled with the 
 ability to measure many locations across many different substrates , 
 large - area measurements are useful for characterizing the self - assembly 
 of psi ( or any protein with direction - dependent rectification ) . additionally , 
 the nondamaging nature of egain makes it a useful tool for investigating 
 sams of psi during the fabrication of a thin - film device , for example , 
 a photovoltaic device , the properties of which are dominated by the 
 average orientation of psi . single - complex measurements provide 
 details that are difficult or impossible to extract from large - area 
 measurements , thus the combination of the two gives a complete picture , 
 capturing the details of transport through individual complexes and 
 the supramolecular structure of the sam . although it is clear 
 that the relative orientation of rci and psi 
 is influenced by the chemistry at the surface of the bottom electrode 
 ( au in our case ) , the absolute orientation of either 
 has not been determined unambiguously . it was observed that rci preferentially 
 platinizes at one side , which was assumed to occur at the more polar 
 ( electron accepting ) side of the complex . despite the lack of direct evidence , this one study has become the 
 reference point for the absolute orientation of rci and , by extension , 
 psi . our data were acquired on sams of psi , which has a different 
 electrostatic profile than rci and therefore does not necessarily 
 orient identically to rci . the first 
 is that charges ( electrons or holes ) take advantage of the electron 
 transport chain or directly traverse it ; both mechanisms involve thermally 
 activated hopping processes . these mechanisms ( it is not clear specifically 
 which ) are used to infer the absolute orientation of rci and predict 
 higher values of r when psi is oriented up . the second 
 mechanism assumes that tunneling charges do not hop through electron 
 transport pathway and the rectification is instead driven by the large 
 dipole moment of the whole psi structure between the luminal and the 
 stromal surface ( i.e. , perpendicular to the substrate when psi is 
 in the up or down orientation ) . van haeringen et al . used linear dichroism 
 to elucidate the dipole moment within the structure of trimeric psi . the direction of the dipole is parallel to the 
 c3-symmetry axis of the protein trimer complex , with positive side 
 of the dipole moment on the luminal side and negative on the stromal 
 side . the dipole effect within psi trimers was also observed on solid - state 
 bulk heterojunction ( bhj ) solar cells with a work function shift . ( there , a monolayer of psi trimers was self - assembled 
 at a metal - oxide electrode rather than au . ) the electric field of 
 this dipole moment can either enhance or oppose the total field created 
 by biasing the electrodes as is shown in figure 3 . although we have no way to observe the absolute 
 up / down orientations of psi directly , we can assign the absolute orientation 
 by determining which mechanism ( the electron transport chain or the 
 internal electric field ) is more likely . first , however , we must establish 
 that cp - afm and egain are in agreement in order to relate our observations 
 to previous reports ( which exist for cp - afm , but not for egain ) . direction 
 of the electrical field ( dashed lines ) that arises from 
 the psi dipole moment within psi - egain devices , which are shown with 
 egain biased positively ( with respect to the normal wiring of egain ) . ( a ) when psi is oriented  up  , the electric 
 field from the applied bias opposes the internal electric field of 
 the psi complexes . the direction of the internal electric field is the same as the applied 
 bias . thus , this mechanism predicts that psi in the down orientation 
 will give higher values of r. although 
 the influence of the native oxide layer on egain has been thoroughly 
 studied on aliphatic sams and has been shown to have a negligible 
 influence on transport properties , it 
 is necessary to confirm that the same holds true for protein complexes . 
 due to the size of complexes of psi , we are able to isolate individual 
 complexes for cp - afm measurements , meaning that the calculation of 
 per - complex resistivity is unambiguous . for egain junctions , we know 
 the density of psi complexes and the measured contact area , ageo , but not the actual contact area , aeff , which is considerably smaller due to the 
 topology of the egain tip thus , we can 
 calculate the number of complexes in ageo , compute the resistance and determine the correction factor to relate ageo and aeff . if 
 the oxide is benign in this study , this correction should be comparable 
 to the values reported for aliphatic sams . for a given egain 
 junction , we considered every oriented psi complex as a resistor in 
 parallel . we calculated the resistance of individual complexes ( ri ) from average values of i from cp - afm measurements at 1 v ( range of 10 to 10 ohms depending on the orientation ) . the 
 resistance , robs , is the total resistance 
 of the circuit and , at v = 1 v , is the reciprocal 
 of the current calculated for an area with n number 
 of complexes , allowing the calculation of n from 
 eq 1 where iegain is the measured current of an egain / ga2o3//psi 
 junction.1 the values of n given by eq 1 are 4.5 and 2.2  10 for 2me and mps , respectively . 
 using these values of n , we calculated aeff from the densities of psi complexes shown in table 1 . from the measured value of ageo , eq 2 gives the overestimation of 
 the area , .2 from this calculation we find   10 , 
 which is consistent with the values of 10 that 
 have been observed for aliphatic sams . thus , using 
 the per - complex conductivity determined by cp - afm and the magnitude 
 of the current obtained from large - area measurements with egain , we 
 arrive at a value of  that is within a factor of 10 of previously 
 reported values . this result shows that the contact resistance associated 
 with the physisorbed electrode - psi interface is comparable for egain / ga2o3 and a pt / ir cp - afm tip and , therefore , that 
 the influence of the oxide layer on charge - transport is negligible 
 ( i.e. , it does not contribute to robs more 
 than a cp - afm tip ) ; cp - afm and egain data are comparable . this results 
 is unsurprising in the context of existing studies on sams of alkanethiolates , but it is necessary to establish the benign 
 nature of the oxide specifically in junctions incorporating proteins . using our computed value of  , we plotted the per - complex 
 current - densities from cp - afm and egain ( figure 4c , d ) . the values of r from egain are in agreement 
 with the values of r from cp - afm data , but figure 4c , d also shows a qualitative agreement between the 
 line shapes . v , j  v curves with respect 
 to the original wiring for cp - afm and egain , respectively ( figure 4a , d ) . top : semilog plots of current and current density 
 versus voltage 
 for junctions measured using cp - afm ( a ) and egain ( b ) for sams of 
 psi on mps ( purple ) and 2me ( black ) the black arrows indicate the 
 orientation of the psi complexes on the surface . these data are plotted 
 according to the normal wiring of each technique ( see the supporting information for details ) . bottom : per - complex j  v curves for sams of psi on 2me ( c ) and mps ( d ) measured by cp - afm 
 ( black squares ) and egain ( green circles ) plotted with respect to 
 the standard wiring of cp - afm ( shown in the insets ) . per - complex values 
 of j for egain were calculated using number densities 
 of psi measured by afm and a correction factor for the difference 
 between the measured and effective area of the egain junctions . while egain junctions can not be 
 scanned past 1 v without 
 precipitating shorts ( from electrostatic pressure ) , cp - afm junctions 
 can be scanned further because the height of the tip is fixed by the 
 instrument rather than the sam of psi . thus , while the cp - afm and 
 egain data are in remarkably close agreement at 1 v , we can not 
 know for certain that they would not diverge at higher potentials . cahen 
 and co - workers have shown , using hmds , that the electrical properties 
 of junctions comprising metalloproteins are affected by the removal 
 of the metal centers . these studies show 
 that tunneling electrons can take advantage of the accessible states 
 of the metal centers . in studies of rci ( not psi ) where rectification 
 is observed , the mechanism is almost always ascribed to the electron 
 transport chain ; however , no evidence is offered to support or refute that hypothesis . previous studies have shown that , when the electron transport chain 
 is deliberately engaged , the rate of electron transfer in rci is higher 
 from p700 to fab ( forward ) than in the reverse direction , which could explain the diode - like behavior seen in the j  v curves . the two 
 most straightforward experiments for establishing the mechanism are 
 removing the electron transport chain and variable - temperature studies . the former is not possible without substantially influencing the structure 
 of rci / psi , and the latter is not possible with the techniques that 
 have been used to study rci thus far . fortunately , the ( remarkable ) 
 robustness of sams of psi in vacuo extends 
 to sufficiently low temperatures to enable variable - temperature studies 
 using microfluidic channels filled with egain in a crossbar configuration . one of the most robust rectifying tunneling 
 junctions comprises ferrocene - terminated sams on ag with egain top - contacts . the mechanism of rectification in these sams is the 
 ( partial ) pinning of the homo of the ferrocene to egain , which pushes 
 it either into or out of resonance with a ag electrode 
 depending on the sign of the applied potential on egain ; when it moves 
 into resonance ( negative potential ) , charges tunneling onto the homo 
 and then hops onto the egain , shortening the effective tunneling distance . this mechanism was proven by arrhenius plots ( ln |j| vs 1/t ) , which clearly show the  freezing 
 out  of the hopping component , leading to the loss of rectification 
 at temperatures below the activation energy of the hopping process . if the mechanism of rectification in psi involves 
 thermally activated hopping processes in the electron transport chain , 
 it should freeze out as well . thermally activated transport processes 
 have also been observed in cp - afm studies of long conjugated molecules and proteins such as azurin , ferritin , and cytochrome at 
 elevated temperatures . we did not collect transport data above room 
 temperature , but at low temperature we observed comparable results . thus , there may be temperature - dependent transport pathways at elevated 
 temperatures , but they do not contribute to the asymmetric transport 
 at room temperature . if the mechanism of rectification in our 
 psi junctions involves 
 the electron transport pathway then it must also involve a hopping 
 process , as the electrons change energy inside the complexes ; moving 
 with the electron transport chain ( downhill ) then leads to higher 
 values of j than against it ( uphill ) at a particular 
 value of |v| . if , however , the mechanism of rectification 
 is the interaction of the applied field with the built - in field of 
 the collective dipole moments of the psi complexes , the process is 
 entirely tunneling ; the rectification arises from 
 the different probabilities of tunneling from left to right and right 
 to left . the former mechanism , therefore , will show a loss of rectification 
 at low temperatures as the hopping processes are frozen out , while 
 the latter will be completely independent of temperature . we 
 fabricated microfluidic devices following literature procedures 
 and acquired j  v traces at different temperatures . an arrhenius plot of ln |j| at 0.50 v for psi 
 on both directing sams is shown in figure 5 ( the raw data are shown in the supporting information ) . the magnitude of r , shown by the difference in 
 the magnitude of j at positive and negative bias , 
 is invariant with temperature . the values of ln |j| are also nearly invariant , showing only a slight perturbation only 
 near room temperature . these data are unambiguous evidence that the 
 mechanism of charge transport through sams of psi on mps and 2me is 
 independent of temperature and , therefore , that the mechanism of rectification 
 does not involve hopping and/or the electron transport 
 chain . from this conclusion , we can ascribe an absolute orientation 
 of psi ; it is oriented  down  ( p700 adjacent to the 
 egain substrate ) on 2me and  up  on mps . thus , the direction 
 of rectification is exactly opposite to the natural flow of electrons 
 through the electron transport chain , which predicts higher currents 
 at positive bias ( with respect to egain ) when p700 is adjacent to 
 the egain electrode . an energy level diagram based on nakamura s 
 et al . it not only predicts that asymmetry will be more 
 pronounced in the up orientation , but that r < 
 1 . note that this diagram is drawn with respect to the normal wiring 
 of egain and is , therefore , backward from the wiring convention of 
 cp - afm . ln |j| at 0.50 v as a function of inverse 
 temperature for psi on directing sams of mps ( triangles ) and 2me ( squares ) . the solid symbols (  ,  ) represent the positive bias 
 ( + 0.50 v ) and hollow (  ,  ) represent the negative bias 
 ( 0.50 v ) . the linearity indicates that the mechanism of charge 
 transport is dominated by tunneling as no temperature 
 dependence was measured . the entirety of the curves for biases from 
 1 to 1 v can be found on the supporting 
 information . the barrier width 
 is defined by the thickness of one oriented psi complex , which is 
 depicted in the  down  orientation with respect to the 
 natural direction of electron flow . the green lines are the frontier 
 orbital energies of the chlorophyll molecules , which are distributed 
 evenly through the thickness of the psi complex . the black lines represent 
 the energies of the electron transport chain and their relative spatial 
 positions . on the basis of the orientation of the electron transport 
 chain , more current should flow when the egain electrode is biased 
 negatively than at the equivalent positive bias . that mechanism would 
 translate into higher values of r when the complexes 
 are oriented  up  ( because this figure is drawn with 
 respect to the wiring of egain junctions ; figure 4 shows the data with respect to the wiring diagram of cp - afm ) . however , in our experiments we observe more current at the positive 
 bias , which supports our dipole moment hypothesis . the distances between 
 cofactors were estimated with the software pymol from a crystal structure 
 of psi taken from the protein data bank ( 1jb0 ) . a common test for tunneling transport is to compare the decay 
 coefficient , 
  , against literature values . this value is obtained from simmons 
 approximation , j = j0e , where d is distance between the electrodes and  is obtained 
 by varying d. this study is not possible with psi 
 because d is fixed by the complex at 6 nm . estimated  by comparing i in the presence 
 and absence of a protein , arriving at values of 0.18 , 0.12 , and 0.27 
  for three different proteins on si surfaces 
 modified with octadecyltrimethoxysilane . using the same analysis , we arrive at a value of 0.16 for psi on 
 2me and 0.08  for psi on mps with d = 60  and values of j taken from 
 table 3 . to gain more experimental insight 
 into the mechanism of rectification , 
 we measured the transport properties of bovine and human serum albumin 
 ( bsa and hsa ) , which have been studied extensively . we observed r  5.5 and 3.0 on 2me and mps , respectively ( see supporting information ) . this observation can 
 only be reconciled using the mechanism of rectification proposed in 
 this paper , given that sams of bsa and hsa show preferential ordering and that they contain only alpha helices , which 
 contribute to a collective dipole moment . it also explains the observation 
 by ron et al . that bsa rectifies on br - terminated si surfaces using 
 hmd electrodes . we have established a clear relationship between the average orientation 
 of psi ( not rci ) in sams and the asymmetry ( rectification ) of current 
 in metal / protein / metal junctions on template - stripped au surfaces 
 modified with directing sam . asymmetry is a useful observable for 
 capturing the complexities of assemblies of large , biological molecules , 
 particularly in combination with single - complex measurements , which 
 are insensitive to collective properties , but which provide details 
 that are missed by large - area methods . the average orientation is 
 affected by the identity of the directing sam used to control the 
 surface chemistry of the substrate . through variable - temperature 
 measurements we have established that 
 the dominant mechanism of charge - transport through sams of psi on 
 au is likely tunneling and that , at the very least , 
 it does not involve thermally activated transport . this observation 
 refutes the hypothesis that rectification is due to the natural direction 
 of the flow of electrons through the transport chain , which involve 
 thermally activated processes , and instead is likely the result of 
 the internal electric field that arises from the collective action 
 of dipoles ( and multipoles ) in the peptide backbone . it opposes the 
 natural direction of electron flow through the electron transport 
 pathway and therefore predicts that 2me preferentially orients psi 
  down  and mps  up  with respect to the 
 direction of the flow of electrons through the transport chain in 
 vivo ; i.e. , with p700 adjacent to the egain substrate . this assignment 
 of the orientations of psi is in agreement with solid - state , thin - film 
 devices comprising psi ( not rci ) that is oriented by modifying the 
 surface of the bottom electrode . we thermally deposited 
 a 120 nm - thick layer of gold ( au 111 ) onto a technical - grade 3 
 silicon wafer supporting a native oxide layer ( si / sio2 ) . we fabricated the substrates by template stripping ( ts ) , where a drop of ultraviolet ( uv)-curable optical 
 adhesive ( oa ) was used to adhere a 1  1 cm piece of glass to 
 a preprepared gold coated wafer . the glass was then mechanically cleaved 
 exposing an ultraflat au surface with a root - mean - squared roughness 
 of 0.3 nm ( as measured using tapping - mode afm ) . next , we immersed 
 the substrates in a solution of 1 mm 2-mercaptoethanol ( 2me ) or sodium 
 3-mercapto-1-propanesulfonate ( mps ) to direct the psi complexes to 
 adopt a down or up orientation ( fb iron  sulfur cluster 
 adjacent or away from substrate ) . the time of immersion was limited 
 to 2 h to avoid the formation of multilayers or aggregates . after 
 this step , were rinsed the substrates with mq water ( mps ) or ethanol 
 ( 2me ) , dried them with nitrogen and incubated them in a previously 
 prepared psi solution . the psi solution 
 consisted of 1:1 in buffer a ( 20 mm hepes ( ph 7.5 ) ; 10 mm mgcl2 ; 10 mm cacl2 ; 500 mm mannitol with 0.05% ddm ( n - dodecyl--d - maltoside ) ) for 2 h. they were 
 then rinsed with mq water and dried with nitrogen . we analyzed each 
 substrate by afm after each fabrication step : on the template - stripped 
 substrate after cleavage , after surface modification , and after incubation 
 in a solution of psi . this was calculated by knowing the diameter 
 of the psi trimer from tem images and the coverage density ( calculated 
 manually ) for specific areas . we obtained the afm images with multimode 
 8 with scanasyst microscope in tapping mode with tesp probes ( bruker ) 
 with spring constant k = 42 nm , resonance frequency f = 320410 khz and 
 tip radius of less than 10 nm . the scan rate and resolution were 1 
 hz and 640 lines / sample , respectively . we studied 
 the conductivity of the immobilized psi on the two orienting 
 monolayers with afm tunneling atomic force microscopy ( tuna ) contact 
 mode with a conducting probe . this mode was applied for electrical 
 characterization of single ( trimer ) psi complex with pt / ir coated 
 si n - type probe ( appnano ) , spring constant k = 0.020.8 
 nm , resonance frequency f = 525 khz and tip radius less than 30 nm with contact resistance 
 of 0.010.025 ohm / cm . the applied force 
 to cp - afm conducting probe on top of psi was started from low and 
 step - by - step increased to reach contacting for i this approach was used for each measurement point with 
 forces of less than 10 nn . we formed 
 the egain electrodes 
 by suspending a drop of egain from a 10 ml syringe on an adjustable 
 stage . using a piezo stepper ( open - loop , 5 nm resolution ) 
 we brought the drop into contact with the sam of psi . the egain adhered 
 to both the needle and the au and by retracting the needle slowly 
 ( ca . 50 ms , we produced conical tips of 
 egain with diameters of 25 m in diameter . we performed the electrical measurements in a custom - built 
 faraday cage using a keithley 6430 sub - femtoamp remote sourcemeter 
 smu instrument . the device was held in place with a spring - loaded 
 gold tip that was isolated from ground . data were obtained from an average of points by 
 sweeping the potential from 1.0 to 1.0 v at a rate of 0.2 
 v / s . to elucidate the transport mechanism of bacterial psi - based devices , 
 we measured the dependence of the electric behavior on temperature . 
 in order to do this , we fabricated encapsulated and addressable devices 
 able to operate in a pressure and temperature controlled setup ( see supporting information ) .
recently , photoactive proteins have 
 gained a lot of attention due 
 to their incorporation into bioinspired ( photo)electrochemical and 
 solar cells . 
 this paper describes the measurement of the asymmetry 
 of current transport of self - assembled monolayers ( sams ) of the entire 
 photosystem i ( psi ) protein complex ( not the isolated reaction center , 
 rci ) , on two different  director sams  supported by 
 ultraflat au substrates . 
 the director sams induce the preferential 
 orientation of psi , which manifest as asymmetry in tunneling charge - transport . 
 we measured the oriented sams of psi using eutectic ga  in ( egain ) , 
 a large - area technique , and conducting probe atomic force microscopy 
 ( cp - afm ) , a single - complex technique , and determined that the transport 
 properties are comparable . by varying the temperatures 
 at which the 
 measurements were performed , we found that there is no measurable 
 dependence of the current on temperature from 0.1 to 1.0 
 v bias , and thus , we suggest tunneling as the mechanism for transport ; 
 there are no thermally activated ( e.g. , hopping ) 
 processes . 
 therefore , it is likely that relaxation in the electron 
 transport chain is not responsible for the asymmetry in the conductance 
 of sams of psi complexes in these junctions , which we ascribe instead 
 to the presence of a large , net dipole moment present in psi .