our initial experiments were 
 focused on establishing for the first time if the in situ generation 
 of an aryl diazonium cation via reduction of p - phenylenediamine 
 with nano2 could be electrochemically coupled to an ito 
 surface via its reduction in situ , forming an arylamine tether layer 
 similar to that previously observed on pyrolyzed photoresist film 
 ( ppf ) electrodes .
two cyclic voltammograms 
 ( cvs ) were performed under the following conditions , a start potential 
 of 0.4 v , a switching potential of 0.6 v , and an end potential 
 of 0.4 v being applied using the ito electrode at a scan rate of 0.1 
 v s. for the second scan , the electrode was held 
 at a potential of 0.6 v for 120 s to ensure all the cation 
 had been reduced .
the resulting grafting scans at ito electrodes of 
 in situ generated aryl diazonium cation can be seen in figure 2a . on scan one
an irreversible reduction peak was 
 observed which can be attributed to the reduction of the diazonium 
 cation at a potential of approximately 0.1 v at the ito surface . 

others have reported the mechanism of surface modification 
 in which the reduction of the diazonium cation leads to the generation 
 of an aryl radical .
the aryl radical then attacks the electrode surface 
 forming a covalent bond between the aryl diazonium and the surface . 

thus , it appears that the reaction mechanism is the same as previously 
 reported at gold and carbon surfaces . on the second 
 scan , at the ito electrode ,
no subsequent cathodic peak is observed 
 for the reduction of the aryl diazonium cation because the film becomes 
 self - limiting .
this is based on the process of multilayer formation 
 by which an increase in thickness leads to a significant increase 
 in the resistance , the potential at the outer surface of the diazonium 
 film is not sufficient anymore to reduce more diazonium cation , and 
 deposition ceases .
( a ) two consecutive cvs were performed ; 
 on solutions of in situ 
 generated diazonium cation . a starting potential of 0.4 v and a switching 
 potential of 0.6 v at 0.1 v s ; 
 prior to the second scan the potential was held at 0.6 v for 
 120 s to confirm surface modification due to the blocking nature of 
 the film .
( b ) typical cyclic voltammograms obtained for solutions 
 of 250 m ferricyanide and 50 mm pbs at modified ( in situ grafting 
 of the diazonium cation ) and unmodified electrodes . to confirm surface modification , electrochemical 
 behavior of the 
 probe ferricyanide was performed and the resulting cvs can be seen 
 in figure 2b .
cvs obtained for 250 m 
 ferricyanide at an unmodified ito results in the observation of a 
 quasi - reversible redox couple with a peak separation of 0.11 v. a 
 cyclic voltammetric scan was then performed for ferricyanide using 
 an arylamine modified ito surface as the working electrode , and no 
 redox couple was observed .
the absence of a defined reversible redox 
 couple can be attributed to the blocking nature of the arylamine film , 
 as previously reported for other electrochemical mediators at diazonium 
 salt modified surface .
high - resolution xps spectra 
 ( figure 3 ) were 
 also acquired on the ito electrodes before and after electrochemical 
 grafting of the arylamine to the ito surface .
high - resolution scans 
 of the n ( 1s ) region show the presence of nitrogen on the arylamine 
 modified ito ; importantly , no n ( 1s ) peak was observed on bare ito , 
 thus confirming the grafting .
the n ( 1s ) spectrum of the arylamine 
 modified ito can be deconvoluted into two peaks with binding energies 
 at 399.9 and 401.2 ev , which are attributed to amino groups ( nh2 ) and protonated ( nh3 ) amino groups .
xps spectra of the n ( 1s ) peak region of ito surface before 
 and 
 after the electrochemical grafting of the arylamine . following the electrochemical and xps confirmation 
 of the efficient 
 electrochemical grafting
, the viability of using chemical self - assembly 
 of swcnts to vertically attach them to the ito surface was demonstrated 
 by atomic force microscopy ( afm ) . after performing electrochemical 
 grafting , the swcnts were covalently tethered to the ito surface via 
 carbodiimide chemistry by forming amide bonds between the amino groups 
 on the surface and the carboxylic acid groups on the swcnts .
for comparison , 
 control surfaces , i.e. , bare ito and electrografted arylamine modified 
 ito , were also analyzed by afm .
a clear difference was observed between 
 the bare ito surface ( figure 4a ) , arylamine 
 modified ito ( figure 4b ) surface , and swcnt 
 modified ito surface ( figure 4c ) .
importantly , 
 the results show bundles of vertically aligned single - walled carbon 
 nanotubes ( vaswcnts ) with a maximum length of 60 nm ( figure 4c ) .
further evidence of surface modification of 
 the corresponding tether layer and swcnts is given by comparing the 
 roughness of each surface with root - mean - squared surface roughness 
 ( rms ) values obtained of 2.73  0.12 , 3.50  0.83 , and 4.87 
  0.92 nm for bare ito , arylamine modified ito , and with covalently 
 coupled swcnts , respectively .
roughness values were obtained by sampling 
 surface areas of 1 m  1 m at three randomly selected 
 locations on each of the three samples .
a one - way anova was performed 
 to show that the mean values were significantly different ( p < 0.000  000  1 ) , demonstrating 
 that with swcnt modification there is significant increase in roughness 
 as would be expected for a heterogeneous surface modified with varying 
 lengths of swcnt .
further evidence for surface modification is provided 
 by cyclic voltammograms of 50 mm ph 7 pbs at the modified surfaces 
 ( figure s1 ) .
as expected , immobilization 
 of the vaswcnts on the ito surface increases the electrode area , resulting 
 in higher charging currents for the swcnt modified ito surface , when 
 compared with bare ito and arylamine modified ito surfaces .
typical afm 
 images of ( a ) bare ito , ( b ) arylamine modified ito , 
 and ( c ) swcnt modified ito .
one of the aims of the research was to enable the 
 efficient uptake 
 of vaswcnts into the intracellular space , while ensuring the swcnts 
 were still attached to the underlying conducting ito surface , to allow 
 for electrochemical sensing inside the cells . to achieve this ,
swcnts 
 were modified with dna by denaturing dsdna by boiling in water for 
 10 min and then placing the electrodes in the dna solution and sonicating 
 for 5 min which causes the produced ssdna to form -stacking 
 interaction with the swcnts . in order 
 to confirm the modification of swcnts with dna , the electrochemical 
 characterization of mb at the varying surfaces was performed .
mb was 
 used for this purpose at it intercalates with dna , giving rise to 
 different electrochemical characteristics when compared to its interaction 
 at normal surfaces .
these studies were 
 also important to allow us to understand the electrochemical behavior 
 of mb at the dna modified surface as it was our intention to use intracellular 
 mb to show proof of principle that intracellular signaling could be 
 achieved using the fabricated surfaces .  
mb has been reported 
 to adsorb to swcnts through charge - transfer 
 and hydrophobic interactions and and 
 to interact with ssdna through electrostatic interactions .
thus , a study was performed to assess the effect on the electron 
 transfer kinetics of mb on the different surfaces .
this was done by 
 initially running a cyclic voltammogram from 0 to 0.4 v at 
 0.1 v s in solutions of 500 m mb ( figure s2 ) . as the eventual monitoring of mb 

will be ascertained in solution using electrodes made in batches , 
 it was important to ascertain their reproducibility .
the mean cathodic 
 peak current obtained for 500 m mb at dna - swcnt modified ito 
 electrode was 22.73 a with a coefficient of variation ( sd / mean 
 multiplied by 100 ) value of 11.1% obtained .
the electrodes were 
 then washed in ethanol and water to remove 
 none adsorbed mb prior to performing voltammograms in pbs , which was 
 performed under the same operating conditions as mentioned previously 
 but at varying scan rates with the following working electrodes : bare 
 ito , arylamine modified ito , and swcnt modified ito , and the latter 
 two surfaces were modified with dna . for the ito surfaces modified 
 with the arylamine no or little adsorption of mb was observed .
this 
 can be attributed to the fact that at ph 7 the amino groups are partially 
 protonated , meaning that they electrostatically 
 repel the charged mb moieties inhibiting mb adsorption . 

parts a(i ) , b(i ) , and c(i ) of figure 5 show 
 typical cyclic voltammograms obtained in pbs at varying scan rates 
 for bare ito , swcnt modified ito , and swcnt and dna modified ito surfaces , 
 respectively , that were previously exposed to mb . as expected for 
 a surface bound process ,
current was observed to be proportional to 
 scan rate at bare ito ( figure 5aii ) , swcnt 
 modified ito ( figure 5bii ) , and swcnt and dna 
 modified ito ( figure 5cii ) electrodes .
if the 
 process was under diffusion control , which would mean that the electroactive 
 species was in solution , then current would be proportional to the 
 square root of scan rate .
mean peak separation potentials ( ep ) obtained at a scan rate of 0.1 v s at bare ito , swcnt modified ito , and swcnt and dna modified ito 
 were 26  4 , 50.7  10.2 , and 77.7  2.08 mv , respectively . 

this data shows the kinetics for electron transfer to the electrode 
 is quickest at the bare ito electrode .
the cyclic voltammograms obtained 
 using the dna modified swcnt ( figure 5c ) demonstrated 
 that with increasing scan rates the ep becomes larger , which is indicative of an electrochemical 
 irreversible process .
we can infer form this behavior that the dna 
 attenuates the rate of electron transfer of mb , and this is due to 
 the electrons having to tunnel through the dna .
additionally , this result also confirms the presence of 
 dna as this decrease in electron transfer rate over the scan range 
 studied is significantly lower at surfaces modified with swcnt only .
typical 
 cyclic voltammograms obtained for solutions of ph 7 . pbs 
 at ( ai ) bare ito , ( bi ) swcnt modified ito , and ( ci ) swcnt and dna 
 modified ito electrodes with absorbed mb at scan rates of 0.02 , 0.05 , 
 0.1 , 0.2 , and 0.4 v s. plots of anodic and cathodic 
 peak currents at different scan rates at ( aii ) ito , ( bii ) swcnt modified 
 ito , and ( cii ) swcnt and dna modified ito . in order to demonstrate that the vaswcnts structures 
 penetrate 
 the plasma membrane , which is 5 nm thick , and have access 
 to the cytoplasm , mouse macrophage cells were stained with mb .
it 
 is well established that mb can enter and is retained inside the cell , and most important , it is a redox active moiety 
 that can be used as a fingerprint to identify if the nanostructures 
 have accessed the cytoplasm .
after performing 
 experiments to establish the electrochemical characteristics of mb 
 at the swcnt and dna modified electrode as described above , we performed 
 transformation assays using ca to enable the cells to 
 take up the swcnts .
dna structures are 
 positioned inside the cell is obtained by observing the characteristic 
 mb redox peaks at approximately 0.233 and 0.128 v 
 ( figure 6a , blue cv ) .
additionally , mb can 
 be observed in the microscopic image in the inset of figure 6 .
a control experiment was performed in an identical 
 surface ( i.e. , swcnt and dna modified ito ) as above , but the cells 
 were not incubated in mb .
this control experiment did not display 
 any redox peaks ( figure 6a , red cv ) , providing 
 further support that the voltammetric peaks observed on the swcnt 
 and dna modified ito surfaces were due to the reduction and oxidation 
 of mb within the cells .
( a ) cvs of mb stained cells on swcnt and dna 
 modified ito ( blue 
 curve ) and swcnt modified ito with no dna modification surfaces ( green 
 curve ) .
cv ( red curve ) of cells not stained on a swcnt and dna modified 
 ito surface .
inset is a typical microscopic image of mb stained cells 
 on the nanostructured electrodes .
it is also demonstrated that in order to observe 
 the electrochemistry 
 of mb the swcnts have to be modified with dna .
a control experiment , 
 wherein swcnts modified ito surfaces in the absence of dna were subjected 
 to the same transformation procedure as above , shows no redox chemistry 
 occurring ( figure 6a , green cv ) .
thus , in order 
 to transform the cells with surface - immobilized swcnts , it was essential 
 that dna was present .
recently , ssdna modified swcnts have been shown , 
 when internalized by an immune cell , not to induce an immune response .
this demonstrates that ssdna modified swcnts 
 are biocompatible which is an important trait for any proposed intracellular 
 sensing system utilizing a immune cell line such as used in current 
 investigations . with reasonable assumptions , it is possible 
 to estimate the area 
 and number of swcnts inside the cells that were attached to an area 
 of 0.123 cm , i.e. , ito electrode area exposed to the electrolyte . 

the magnitude of the peak current in the cv for swcnt and dna modified 
 ito ( figure s2 , light blue curve ) provides 
 information regarding the electroactive area of the swcnt 
sevcik equation:1where ip is the 
 peak current ( 2.66  10 a ) , n ( equal to 2 ) is the number of electrons involved in the mb redox 
 reaction ,  is the scan rate ( 0.10 v s ) , a is the electroactive area to be determined ( cm ) , d is the diffusion coefficient of mb in solution 
 ( 4.0  10 cm s ) , and c is the bulk 
 concentration of the mb solution ( 5.0  10 mol cm ) .
the electroactive area , a , was calculated and was found to be 0.111 cm . by knowing 
 the relationship between peak current ( 2.66  10 a ) and electroactive area ( 0.111 cm ) of the swcnt  dna 
 system , it is now possible to calculate the electroactive area responsible 
 for the peak current obtained for swcnt based electrodes that the 
 cells internalized ( 1.17  10 a , figure 6 , blue curve ) .
thus , the value of the electroactive 
 area of swcnt intracellular electrodes became 4.88  10 cm . by considering that the maximum length of swcnts 
 is up to 60 nm as demonstrated by the afm images and the distance 
 between the cells membrane and substrate is between 20 and 40 nm
, we still have a length of swcnt of up to 2040 
 nm that can cross into the cytoplasm .
taking into account that the 
 cell membrane is 45 nm , 1535 
 nm of the swcnt can be inside the cell .
if we assume that the diameter 
 of all swcnts is 1 nm , a surface area per swcnt between 4.79  
 10 cm ( i.e. , swcnts with an intracellular 
 length of 15 nm ) and 1.11  10 cm ( i.e. , swcnts with an intracellular length of 35 nm ) can be in contact 
 with cytoplasm .
if we then divide the total electroactive area inside 
 the cells ( 4.88  10 cm ) by the 
 surface area per swcnt ( 4.79  101.11 
  10 cm ) , we can derive that 
 approximately between 4.41  10 and 1.02  10 swcnts have been taken up by cells that were attached to 
 an ito electrode area of 0.123 cm .
in summary , we have shown that short vaswcnts 
 coupled to an arylamine tether layer on an ito substrate were vertically 
 aligned .
these were further modified with dna , which enables cells 
 to naturally take up carbon nanotubes , and these remained covalently 
 bound to the surface of the electrode via the arylamine tether layer . 

once the swcnts had entered the cell , intracellular communication 
 was established by sensing the electrochemical mediator methylene 
 blue .
establishment of a generic entry mechanism for swcnts that remain 
 covalently bound to a substrate is of fundamental importance and will 
 facilitate future sensing platform that may be used for unprecedented 
 spatial and temporal electrochemical intracellular biosensing .
phosphate buffered saline was prepared by 
 mixing solutions of 50 mm k2hpo4 and 50 mm kh2po4 to give the desired ph 7 and adding an appropriate 
 amount of potassium chloride to give a final concentration of 0.1 
 m saline in 50 mm phosphate buffer .
methylene blue stock solution was prepared at a concentration 
 of 10 mm in sterile pbs .
initially , ito deposited on corning 
 low alkaline earth boro - aluminosililicate glass with indium tin oxide 
 coated on one surface ( resistivity = 2025  ) purchased 
 from delta technologies limited was rinsed with hplc grade ethanol 
 and then ultrahigh purity h2o , exposed to uv light for 
 an hour , and then rinsed in acetone and isopropyl alcohol .
the strategy 
 for preparation of functionalized swcnts covalently attached to ito 
 was based on using an electrografted tether layer which then acted 
 as an anchor for the swcnts .
ito samples were modified in situ with p - phenylenediamine ( ap ) using a similar reported method 
 for carbon surfaces . at room temperature , 
 a 10 mm p - phenylenediamine solution was rapidly added 
 to 1 m nano2 equivalent solution and allowed to react for 
 3 min .
the reaction solution was then poured into the electrochemical 
 cell , and the resultant diazonium cation was electrografted to the 
 ito scanning from 0.4 to 0.6 v followed by fixed potential 
 deposition at 0.6 v for 2 min and finally a further scan from 
 0.4 to 0.6 v to confirm passivation ( and hence grafting ) of 
 the electrode .
uncut swcnts ( nanolab , inc . ) were acid - treated 
 by adding 25 mg to 27 ml of a 3:1 mixture of concentrated h2so4 and hno3 and sonicating for 10 h. following 
 sonication , the contents were poured into 500 ml of distilled water 
 and left to settle overnight .
the swcnts were then filtered through 
 a 0.22 m hydrophilic pvdf filter ( millipore ) under suction , 
 with washing until the rinse water was close to ph 7 .
suspensions of cut 
 swcnts were prepared by sonication of dried swcnts mats in dimethyl 
 sulfoxide ( dmso ) .
swcnts were coupled to arylamine modified ito samples 
 by submerging modified ito samples in a 0.2 mg ml dmso suspension of cut swcnts containing 0.5 mg ml dicyclohexyl carbodiimide ( dcc ) .
the reactants were sonicated for 
 30 min and then heated to 65 c for 24 h in a closed cell .
after 
 preparation , swcnt modified electrodes were sonicated in acetone for 
 2 min and isopropyl alcohol for 10 s and finally rinsed in milli - q 
 water .
swcnt were modified with dna on the surface of the ito 
 electrodes 
 by using an adapted method of that described by zheng et al .
first , dna , obtained from salmon sperm , was 
 denatured via placement of 2 mg ml dna into boiling 
 water for 30 min .
an aqueous solution of ssdna ( 1 mg ml ) was then prepared , and swcnt modified ito sample was immersed and 
 sonicated for 5 min to allow -stacking interactions 
 to form between the dna and swcnts .
all electrochemical studies were carried 
 out with a gamry 600 potentiostat and data acquisition software ( gamry 
 electrochemistry software version 5.61a ) and a three - electrode cell 
 consisting of a saturated calomel reference electrode , pt counter 
 electrode , and then the working electrode of either bare ito , ito 
 modified with an arylamine tether layer , an ito modified with swcnt , 
 or ito modified with swcnt and ssdna .
the electrochemical area was 
 controlled via use of a o - ring with a diameter of 4 mm .
cyclic 
 voltammetry was performed to confirm grafting of in situ produced 
 diazonium cation by running scans of 250 m solution of ferricyanide 
 in 50 mm pbs ( 0.1 m kcl ) from a starting potential of 1.0 v and a 
 switching potential of 0.4 v and an end potential of 1.0 v 
 at arylamine modified and unmodified surfaces .
cyclic voltammetric 
 studies were performed to characterize the electrochemical behavior 
 of mb at the various surfaces .
the operating conditions for the cyclic 
 voltammetry was a start potential of 0 v with a switching potential 
 of 0.4 v at a scan rate of 0.1 v s. controls 
 of pbs were performed at each surface and 500 m solution of 
 methylene blue was utilized . for surface adsorption studies
, the scan 
 rate was varied between 0.02 and 0.4 v s. prior 
 to performing any scans on the electrodes exposed to mb , surfaces 
 were rinsed in ethanol and water .
peak currents were obtained by subtracting 
 the background using linkfit software and measuring the cathodic peak 
 current of three replicates .
afm images were recorded using 
 the nanowizard ii atomic force microscope 
 ( jpk instruments , germany ) .
the images were obtained with cantilever 
 model rtesp part mpp-11100 - 11 ( veeco ) operating in tapping mode at 
 a frequency of 345384 khz ; 10 m  10 m 
 scans were recorded at 1 m s. postprocessing 
 of afm images was performed on version 3.6 nanowizard image processing 
 software and included digital leveling of the images and roughness 
 calculations .
surface characterization was performed on three samples 
 of each surface which included bare ito , arylamine modified ito , and 
 swcnt modified ito .
x - ray photoelectron 
 spectroscopy ( xps ) spectra were obtained on the vg escalab 250 instrument 
 based at the leeds epsrc nanoscience and nanotechnology research equipment 
 facility ( lennf ) at the university of leeds , uk .
xps experiments were 
 carried out using a monochromatic al k x - ray source ( 1486.7 
 ev ) and a takeoff angle of 90. a high - resolution scan of n ( 1s ) 
 was recorded using a pass energy of 20 ev at a step size of 0.05 ev . 

sensitivity factors used in this study were the following : 
 n ( 1s ) , 1.73 ; c ( 1s ) , 0.298 ; o ( 1s ) , 0.711 ; in ( 3d 5/2 ) , 13.32 ; in 
 ( 3d 3/2 ) , 9.22 ; sn ( 3d 5/2 ) , 14.8 ; sn ( 3d 3/2 ) , 10.25 .
raw 264.7 cells ( 2.5  10 cells ) were seeded in a 75 cm flask containing 12 ml 
 of dmem ( 10% fbs , 1 ml of penicillin / streptomycin , 2.4% glutamate , 
 and 2.4% hepes ) .
the waste was decanted , and the cell pellet was resuspended 
 in 1 ml of dmem .
subsequently , the cells were counted , giving 13.0 
  10 cells / ml . this was further diluted , yielding 
 a solution of 1  10 cells / ml .
an aliquot of 10 mm 
 stock solution of mb was added to cells , giving a final assay concentration 
 of 25 m .
the cells were incubated for 30 min with mb and then 
 washed three times in pbs in order to remove mb in solution .
the cell 
 pellet was then resuspended in dmem to give final cell count of 1 
  10 cells / ml
. 2 ml of dmem and 2 ml of hbs solution 
 were then aliquoted into a 6-well plate ( 12.5 l of 2  
 hbs 8.0 g of nacl , 0.37 g of kcl , 201 mg of na2hpo47h2o , 2.0 g of glucose , 5.0 g of hepes/500 
 ml , ph 7.05 ) , 65 l of 1 m calcium chloride and 1 ml of the 
 1  10 cell suspension as added to a 6 well titer 
 plate containing the ito - swcnt modified with dna in the presence of 
 calcium and incubated for 13 h. the electrodes were rinsed with dmem , 
 and electrochemical analysis was performed in pbs .
 the ability to monitor intracellular events in real time 
 is paramount 
 to advancing fundamental biological and clinical science . 
 we present 
 the first demonstration of a direct interface of vertically aligned 
 single - walled carbon nanotubes ( vaswcnts ) with eukaryotic cells , raw 
 264.7 mouse macrophage cell line . 
 the cells were cultured on indium 
 tin oxide with vaswcnts . 
 vaswcnts entered the cells naturally without 
 application of any external force and were shown to sense the intracellular 
 presence of a redox active moiety , methylene blue . 
 the technology 
 developed provides an alluring platform to enable electrochemical 
 study of an intracellular environment . 
Results and Discussion
Conclusions
Experimental Section