medical diagnostics 
 stands to benefit from the development of cost - effective 
 and reliable protein detection methods . detection of protein  protein 
 or protein  ligand interactions , which are vital processes in 
 living organisms , deepens the understanding of associated biological 
 processes and provides invaluable tools to diagnose many diseases . 
 in this context , the suitability of field - effect transistors ( fets ) 
 based on one - dimensional nanomaterials , such as nanowires and single - walled 
 carbon nanotubes ( swnts ) , as versatile and label - free electronic biosensors 
 has been thoroughly studied . their ease 
 of integration into next - generation electronic circuits , coupled with 
 dimensions on the size order of individual molecules , results in promising 
 potential applications in diagnostics . when testing 
 a biosensor s feasibility , well - known biosystems , such as the 
 hybridization of single - stranded nucleic acids or the binding of streptavidin or avidin to biotin play an important role . usually , one of the system s 
 components is immobilized onto the sensitive area of the sensor as 
 a probe , while the other is used as a target analyte . both of these 
 testing systems utilize properties that are exceptional in nature , 
 taking advantage of dna s high charge and the high binding 
 affinity between streptavidin and avidin to biotin ( kd is in the order of 10 m ) . these strong interactions have helped immensely 
 in understanding the underlying sensing mechanisms and restrictions 
 of fet sensors in detail . the dominant sensing mechanism for functionalized 
 swnt fets is commonly accepted as electrostatic gating due to the 
 presence of charged moieties in the proximity of the nanotube s 
 surface , leading to a shift of the threshold voltage of the transistor . while this effect is largely responsible for 
 the ability to perform label - free sensing , devices are limited in 
 terms of the electric double layer forming a so - called debye screening 
 length , rendering sensing in physiological conditions challenging . in this article , we address the need to develop alternative 
 biosystems 
 closer to real demands of fet sensors , which can be used to optimize 
 the sensor for real - life applications , such as point - of - care ( poc ) 
 testing . for this purpose , we used the captavidin  biotin system , 
 which offers a ph - tunable binding affinity with strong affinity occurring 
 at ph 4 and complete dissociation of the protein  ligand complex 
 occurring at ph 10 . hence , these biotinylated 
 devices can be regenerated by washing with ph 10 buffers , rendering 
 this system interesting for engineering biosensors capable of selective 
 protein detection . additionally , the strong interactions 
 of the biotin-(strept)avidin system , widely used in biotechnology , were utilized with our devices to create protein 
  fingerprint  sensors by studying swnt fet response 
 under varying ph values . finally , captavidin experiments were also 
 performed with gold nanoparticle ( aunp ) decorated devices , offering 
 the possibility for complementary surface enhanced raman spectroscopy 
 ( sers ) measurements . briefly , interdigitated electrodes ( with channel 
 length of 10 m ) were patterned via a standard photolithography 
 procedure on silicon substrates ( 200 nm sio2 , 30 nm ti , 
 100 nm au ) . p2-swnts from carbon solutions , inc . ( purity > 90% ) 
 were 
 dispersed in n , n - dimethylformamide ( dmf ) and deposited 
 as the active layer between the predefined electrodes via alternating 
 current dielectrophoresis . the chips 
 were placed onto a ceramic dual in - line package fitted with a liquid 
 compartment affixed by polydimethylsiloxane ( pdms ) . electrical characterization 
 in this work was conducted by recording transfer curves at a fixed 
 source - drain bias of 50 mv and sweeping the gate potential from 0.6 
 v to + 0.6 v. the transistors were operated in electrolyte - gate mode 
 with a ag / agcl electrode immersed in the buffer solution ( figure 1a ) . ( a ) schematic illustration of an electrolyte - gated 
 single - walled 
 carbon nanotube field - effect transistor ( swnt fet ) . transfer 
 curve 1 was taken before exposure to the protein , 2 after incubation 
 with 140 nm captavidin , and 3 after 15 min exposure to ph 10 buffer , 
 which is known to reverse the biotin / captavidin binding . ( c ) normalized 
 sensor response ( i0ip)/gm at different buffer 
 ph as a function of captavidin concentration . ( d ) effect of debye 
 screening length on sensor response to 140 nm captavidin in ph 4 buffer 
 with varying concentrations of kcl . aunp - swnt hybrid devices were fabricated on chip via bulk 
 electrolysis 
 in a three - electrode configuration . the nucleation of gold nanoparticles 
 of varying size from chloroauric acid ( 1 mm aucl3 in 0.1 
 m hcl ) onto the swnts was realized with a nucleation potential fixed 
 at 0.4 v and varying pulse durations ( 1040 s , further 
 details are supplied in the supporting information ) . for the detection of biotin - binding proteins , the swnts 
 were functionalized 
 with pyrene - biotin ( p  b ) by incubating the devices in a 50 
 m p pyrene 
 is known to adhere onto carbon nanotube sidewalls via  
 stacking interactions and is well established for functionalizing 
 carbon nanotubes in biosensors . captavidin experiments with aunp decorated fets were conducted after 
 incubation with 50 m p  b and 50 m ( )--lipoic 
 acid  biotin ( la - b ) for 42 h in methanol . the functionalized 
 devices allow for reproducible results even after extended storage 
 ( see supporting information ) . for 
 protein experiments , a universal buffer ( britton - robinson ) 
 was used and adjusted to the desired ph value by koh titration . the compositions of the buffers were adjusted to the requirements 
 of each experiment as detailed in the supporting 
 information . biotin - binding proteins captavidin , neutravidin , 
 and streptavidin were purchased from life technologies . further 
 information regarding the device fabrication , functionalization , 
 characterization , additional sem images of aunp decorated fets , as 
 well as details on the synthesis of the functionalization molecules 
 are provided in the supporting information . captavidin is a commercially available , 
 tyrosine modified avidin , 
 which offers the advantage of a ph - dependent binding affinity toward 
 biotin with its highest association constant of > 10 m at ph 4 and complete dissociation occurring 
 at ph 
 10 . this system has been already successfully 
 exploited using surface plasmon resonance biosensors by garca - aljaro 
 et al . here , we demonstrate that the 
 biotinylated swnt fet surface can be regenerated after a ph 10 washing 
 step by monitoring the transfer characteristics before and after protein 
 incubation as well as after ph 10 device regeneration . as previously 
 demonstrated , protein sensing may suffer from parasitic signals that 
 can eventually occur from protein attachment onto the gate electrode . thus , we avoided any contact of the electrode 
 with solutions containing the protein by removing the electrode from 
 the fluid chamber during protein incubation . a transfer curve ( i d vs vg ) of the device was 
 taken in ph 4 buffer ( 1 ) before incubation in 140 nm captavidin for 
 15 min . after thoroughly rinsing the device with ph 4 buffer to ensure 
 the removal of all excess protein from the solution , a second i d vs vg transfer characteristic was recorded ( 2 ) . bound protein was then removed with a ph 10 washing step ( detailed 
 in supporting information ) and then transfer 
 curve 3 was recorded . after protein incubation , a threshold 
 voltage shift of about 20 
 mv toward negative voltages was observed . this shift was entirely 
 reversed after the ph 10 washing step , indicating a complete dissociation 
 of the biotin - captavidin complex . b functionalized swnt fets 
 is repeatable multiple times and was successfully used for various 
 experiments . control experiments were performed to verify that pristine , 
 unfunctionalized devices do not show this recovery after washing ( supporting information figure s4c ) . using 
 this biosystem , further experiments were performed to gain 
 information about binding strength and the effect of debye screening 
 length on the sensor s response . to probe the ph - dependent 
 binding affinity of the biotin - captavidin complex , data obtained from 
 four different transistors were analyzed and the results are shown 
 in figure 1c . in the graph , normalized sensing 
 response ( ( |io  ip|)/gm)|vd is plotted as a function of captavidin concentration , 
 allowing comparison between devices by compensating for device - to - device 
 variations . i0 , ip , and gm are extracted from transfer 
 curves , where i0 is the drain current 
 at vg = 0.6 v before protein incubation , 
 ip is the drain current at the same potential after protein 
 incubation , and gm is the maximum of the transconductance 
 id/vg . using previously published analytical models to determine 
 the dissociation constant of captavidin at ph 4 and ph 5 , data were 
 fitted according to eq 1.1 in this equation , [ b]max is the 
 maximum surface density of binding sites on the surface of the carbon 
 nanotubes , c0 is the analyte / channel capacitance , 
 and qp is the charge from the protein . our sensing protocol differs significantly from previous work utilizing 
 streptavidin and other proteins with extremely high binding affinities , 
 wherein sensors can not be reused after a protein binding experiment . here , captavidin was released from the sensor s surface after 
 incubation with a certain protein concentration by a ph 10 washing 
 step and devices could be reused to obtain data for subsequent protein 
 concentrations . as swnt fets are intrinsically sensitive toward changes 
 of the electrolyte s ph , all transfer curves were taken at 
 ph 4 , however the 15 min protein incubations were performed at ph 
 4 or ph 5 . at ph 4 and ph 5 , dissociation 
 constants of 43  7 and 308  170 nm were obtained , respectively . as previously mentioned , experiments addressing the effect of ionic 
 strength on protein sensing can also be performed using the captavidin biotin 
 system . by consecutively sensing the response upon incubation of 140 
 nm captavidin in ph 4 buffer with varying kcl concentrations ranging 
 from 0.25 mm to 50 mm , the response of four transistors was analyzed 
 and the results are depicted in figure 1d . 
 the graph shows the normalized response as a function of the debye 
 screening length ( ld ) , which was calculated 
 by solving eq 2 for monovalent ions.2here , lb is the 
 bjerrum length ( 0.7 nm ) and i and zi the density and the valence 
 of ion species , i. these results are in agreement 
 with the work of sorgenfrei et al . on fet biosensors , in that the 
 sensing response depends exponentially on the ionic strength of the 
 electrolyte . furthermore , we find that 
 the response drops significantly in a ld regime on the 
 order of the hydrodynamic diameter of avidin at a distance from the 
 nanotubes surface , which is given by the length of the functionalization 
 molecule . the p  b length in the 
 lowest - energy conformation estimated using molecular modeling ( spartan 
 10 software ) was 2.7 nm . additionally , ph - dependent measurements 
 with two other biotin - binding 
 proteins , neutravidin and streptavidin , were performed . for each ph 
 value , transfer curves were recorded before and after protein incubation . all protein incubations were conducted in 10 mm pbs buffer ( ph 7 ) 
 for 15 min . in contrast to measurements with captavidin , however , 
 sensors can not be regenerated after binding to biotin ; thus , each 
 measurement requires a new transistor . the curves in panels a and 
 b were collected for biotinylated devices at buffer ph s of 
 3.4 and 6.3 , respectively . we observed that the threshold voltage 
 shift after neutravidin incubation depends on the buffer ph ( experiments 
 with streptavidin reveal the same trend , thus transfer characteristics 
 are not shown ) . for the experiment conducted at ph 3.4 , the threshold 
 voltage shifts toward more negative gate voltages and for ph 6.3 the 
 threshold voltage shifts in the opposite direction . this phenomenon 
 does not occur for experiments with unfunctionalized swnt fets as 
 shown in figure 2c . the id  vg measurements in figure 2c show an overall decrease in device conductance 
 and a slight tilt of the curve after protein adsorption , indicating 
 that the mobility of the device is affected upon protein adsorption , 
 a phenomenon which does not appear if a spacer provided by the functionalization 
 prevents unspecific protein adsorption onto the sidewalls of the carbon 
 nanotubes . transistor characteristics of a biotinylated swnt fet before ( black ) 
 and after ( red ) neutravidin adsorption , measured at ph 3.4 ( a ) and 
 at ph 6.3 ( b ) . ( c ) transfer characteristics of an unfunctionalized 
 device before ( black ) and after ( red ) neutravidin adsorption measured 
 at ph 6.5 . ( d ) normalized sensor response ( i0  ip)/gm of pyrene - biotin - functionalized swnt fet toward neutravidin 
 ( blue ) and streptavidin ( gray ) under different buffer concentrations 
 as a function of buffer ph . the protein response was measured in 1.62 
 mm buffer ( triangles ) and 0.80.9 mm buffer ( circles ) concentrations . to further investigate this phenomenon , we conducted the 
 same experiments 
 for a broad range of ph values for neutravidin and streptavidin ; the 
 results thereof are presented in figure 2d . the data points of this graph are calculated by taking the normalized 
 currents at a gate potential of 0.6 v from the transfer curves 
 before and after protein binding . the experiments for both proteins were conducted at 
 two different buffer dilutions which resulted in buffer concentrations 
 of 1.62 mm ( triangles ) and 0.80.9 mm ( circles ) . irrespective of the parameters applied , a transition 
 from a negative to a positive transistor response can be observed , 
 which is best described by the intercept of the fitted curves with 
 the horizontal , which is plotted as dashed - dotted gray line . the intercept the measurements 
 at the higher buffer concentration yielded a pzr at ph 4.7 for neutravidin 
 ( blue ) and a pzr at ph 6.2 for streptavidin ( gray ) . at the lower buffer 
 concentration , a pzr at ph 5.1 for neutravidin and a pzr at ph 4.2 
 for streptavidin were obtained . in general , we suspect that the transition 
 from negative to positive response is associated with the zeta potential 
 of the proteins , which is the potential at the hydrodynamic shear 
 boundary . in particular , for proteins , surface charges arise due to 
 ph - dependent ionization of the acidic or basic side groups of amino 
 acids located at the surface . in this regard , the zeta potential , 
 which can be positive or negative depending on the ph value , gives 
 information on a protein s electric landscape in solution . at the transition point from positive to negative 
 charge , there is a point of net neutral charge , or the isoelectric 
 point ( iep ) . the zeta potential and the iep are usually determined 
 by measuring the particle motion under an applied electric field in 
 solution for various buffer ph . similarly , a ph dependent transition 
 from a positive to a negative transistor response , including a point 
 of zero response , should be also detectable with a field - effect transistor . namely , if a positively charged particle approaches 
 the vicinity of the surface of a swnt fet , the net charge of this 
 particle will cause a threshold voltage shift toward the positive x - axis , which results in i0  ip < 0 and vice versa for 
 negatively charged particles . neutravidin is an engineered protein 
 with an almost neutral iep of 6.3 as stated by the provider ( invitrogen ) . various ieps for streptavidin are reported throughout the literature 
 and range from roughly 5 to 7.5 . however , 
 those values were obtained in solutions with higher ionic strength 
 than those used in this work . hence , to compare our data with previous 
 results , experiments with neutravidin and streptavidin under different 
 ph values were conducted using a zeta potential analyzer ( details 
 and corresponding data are provided in the supporting 
 information ) . neutravidin revealed an iep of 4.8 for both buffer 
 concentrations and streptavidin yielded an iep of 5.1 in 1.6 mm pbs 
 and of 4.8 in 0.8 mm pbs ( supporting information figure s6 ) . for neutravidin , the values obtained from the two independent 
 experiments are in good agreement . however , the discrepancy between 
 the zeta analyzer and the fet experiments is significant for streptavidin . 
 in both experiments , the iep and pzr depend on the electrolytic conditions 
 wherein a shift toward more acidic ph for lower buffer concentrations 
 is observed . however , the shift is much greater for the fet measurements , 
 possibly because of changes in salt concentration having an effect 
 on the equilibrium of charges in basic and acidic amino acids , which 
 can modify the protein s iep . however , predicting the pzr obtained 
 with an fet for a specific protein and at a certain ionic strength 
 requires exact knowledge of the surrounding potential and charge distribution 
 of the protein . although the observed behavior for our two model proteins 
 can not be considered universal because of well - known batch - to - batch 
 variations ( a broad spectrum of isoelectric points is reported for 
 streptavidin ) , we believe that such zeta - potential - like 
 swnt fet measurements can provide a new tool to differentiate between 
 proteins and thus give rise to new cost - effective methods applicable 
 in fields such as protein engineering . there is an increasing 
 interest in surface enhanced raman spectroscopy 
 ( sers ) for applications in chemical and biological sensing , due to 
 its unique sensitivity with regards to molecular fingerprinting . surface enhanced raman spectroscopy can be realized by decorating 
 carbon nanotubes with gold nanoparticles directly on chip by electrodeposition 
 from a aucl3 solution . to assess 
 the feasibility of this approach for solution processed swnt fets , 
 we utilized aunp - swnt hybrid devices , the fabrication of which is 
 detailed in the experimental section . in figure 3a , a scanning electron microscope image of a gold 
 decorated device ( deposition parameters 0.4 v for 10 s ) reveals 
 a distribution of particle sizes ranging from 50 to 200 nm . the particle 
 sizes obtained from the applied parameters for this device are 50 
 to 200 nm . ( b ) raman spectra of gold nanoparticle decorated swnts 
 using varying deposition times of bulk electrolysis ( deposition voltage 
 0.4 v ) . ( c ) transfer characteristics of a swnt fet recorded 
 before and after functionalization with gold nanoparticles . ( d ) transfer 
 characteristics recorded for captavidin detection with a la - b functionalized 
 device . ( 1 ) transfer curve was taken before exposure to captavidin , 
 ( 2 ) after incubation with 140 nm captavidin , and ( 3 ) after 15 min 
 exposure to ph 10 buffer . ( e ) raman spectra of gold nanoparticle - decorated 
 swnts functionalized with la - b after incubation with captavidin and 
 after ph 10 washing . peaks unique to the protein can be discerned 
 after incubation which disappear upon rinsing . the enhancement of the swnt sers 
 signal is proportional to both the size of the nanoparticles as well 
 as the variation in interparticle distances , and thus on the duration 
 of the deposition pulse . we do not observe 
 a shift in the frequency of the enhanced modes , from which we conclude 
 that electromagnetic enhancement plays the predominant role of sers 
 here . electrical characterization of an swnt fet before and after 
 gold deposition is shown in figure 3c . the 
 rise in conductance of the devices after gold deposition can be attributed 
 to additional metallic pathways between the source and drain electrodes 
 as well as the direct charge transfer between aunps and swnts . for protein sensing , ( )--lipoic 
 acidbiotin 
 ( la - b ) molecules were synthesized to enable covalent biotinylation 
 of the gold particles . b to prevent signals from unspecific protein adsorption 
 onto the pristine nanotubes . in figure 3d , transfer curves upon similar to the experiments without gold nanoparticles , 
 we observe a 20 mv shift of the threshold voltage . in addition to 
 electrostatic gating , the threshold voltage shift may be explainable 
 by a modulation of aunp - swnt schottky barriers due to the presence 
 of the protein . after 
 attachment of captavidin , spectra show clear protein signals , specifically 
 the 1240 cm peak , indicative of -sheet 
 protein structures , which disappear after 
 ph 10 washing ( figure 3e ) . for sers to occur 
 the analyte species must be in close proximity to the sers substrate 
 surface , in this case the aunps . this raman signal enhancement is 
 generally aided by specific binding such as dna hybridization , protein - antibody interactions , and avidin  biotin coupling . advantages of using sers in combination with fet sensors include 
 its ability to perform dry measurements , whereas the ionic strength 
 of electrolyte solution and background signal drift may influence 
 the fet device ; sers provides molecular fingerprints allowing for facile differentiation of proteins ; last it 
 is a nonelectrical method , which is immune to parasitic signals from 
 analyte - electrode interactions , and complements fet characterization . this type of dual mode measurement could be extremely useful for development 
 of future biomedical applications . hereby , we introduced 
 captavidin , a tyrosine modified avidin , as a useful protein to engineer 
 and optimize fet biosensors due to its reversible binding to biotin . 
 using this biosystem , we were able to probe the dissociation constant 
 of captavidin at two different ph values and to demonstrate that the 
 sensor signal depends on the debye screening length . furthermore , 
 we were able to differentiate between two different biotin - binding 
 molecules , streptavidin and neutravidin , by their ph - dependent sensor 
 response . additionally , swnt fets decorated with gold nanoparticles 
 were shown to be a promising platform providing a supplemental surface 
 enhanced raman spectroscopy substrate useful for the detection of 
 proteins .
we 
 report on the reversible detection of captavidin , a tyrosine 
 modified avidin , with single - walled carbon nanotube ( swnt ) field - effect 
 transistors ( fets ) noncovalently functionalized with biotin moieties 
 using 1-pyrenebutyric acid as a linker . 
 binding affinities at different 
 ph values were quantified , and the sensor s response at various 
 ionic strengths was analyzed . 
 furthermore , protein  fingerprints  
 of neutravidin and streptavidin were obtained by monitoring their 
 adsorption at several ph values . 
 moreover , gold nanoparticle decorated 
 swnt fets were functionalized with biotin using 1-pyrenebutyric acid 
 as a linker for the cnt surface and ( )--lipoic acid linkers 
 for the gold surface , and reversible captavidin binding is shown , 
 paving the way for potential dual mode measurements with the addition 
 of surface enhanced raman spectroscopy ( sers ) .