our device incorporates three key 
 elements : patterned polymer brushes 
 to present selected haptenic groups ; cofactors required for acwop ; 
 and components for electrochemical detection and quantification of 
 h2o2 ( figure 1 ) .
details 
 about the materials and methods used and additional control experiments 
 are given in the supporting information .
a fundamental feature of our device is the use of poly(oligoethylene 
 glycol methacrylate ) ( poegma ) polymer brushes ( figure 2a ) for anchoring a variety of hapten groups and for preventing 
 nonspecific adsorption of other biomolecules that may be present in 
 the test sample .
oeg moieties are known to be resistant to protein 
 adsorption and have long - term stability .
this is due to the dense packing of neighboring chains which results 
 in an increased entropic force and drives the brushes into a stretched 
 state at high grafting densities to yield effective resistance to 
 nonspecific binding . to produce the necessary 
 high grafting density , we employed atom transfer radical polymerization 
 ( atrp ) methods to grow brushes on either a silicon wafer chip or a 
 gold - plated quartz crystal microbalance ( qcm ) crystal . by functionalizing the initiator end with either 
 a silane or thiol group ,
polymer brushes have the capacity 
 to be modified with a broad range of haptens for corresponding detection 
 of antibodies with a broad range of specificities . for initial 
 development and optimization of this platform , we used 
 2,4-dinitrophenyl ( dnp ) groups as a model hapten .
this well - characterized 
 hapten binds specific anti - dnp antibodies of several classes and species 
 and can be conjugated to the ends of the brushes through a one - step 
 process ( figure 2a ) .
the functionalized polymer 
 brushes ranged in thickness from 13 to 35 nm , measured via ellipsometry , 
 and two methods were utilized to confirm the presence of dnp groups . 

first , fluorescence imaging with fluorescently labeled anti - dnp igg 
 antibodies verified the presence of the dnp groups when compared to 
 a sample of unfunctionalized brushes ( supporting 
 information figure s1 ) .
second , dnp is electroactive , allowing 
 measurement of its surface coverage through cyclic voltammetry by 
 means of the reduction of nitro groups to the corresponding hydroxylamine 
 ( figure 2b ) .
the surface coverage of dnp was 
 typically on the order of 10 mol / cm , and as high as 1.5  10 mol / cm .
inset : cyclic 
 voltammogram of hydroxylamine formed upon reducing dnp . supporting 
 electrolyte , 0.1 m h2so4 ; sweep rate , 100 mv / s . 
 ( c ) frequency response of qcm crystal platform in pbs , ph 7.2 , containing 
 1 mg / ml bsa solution at 25 c .
rat anti - dnp igg antibody solution 
 ( resultant concentration 11 nm ) was added at 9000 s. ( a , b ) grid patterns of 150 m wide lines surrounding 
 300 
  300 m square areas on silicon ( a ) and gold 
 electrode of a qcm crystal ( b ) .
solid lines 
 are for qcm crystal platform immersed in 0.5 mm [ ru(v - bpy)3](pf6)2 , showing electropolymerization of photosensitizer 
 upon reduction . dashed lines are for an electropolymerized layer of 
 [ ru(v - bpy)3 ] in fresh solution . supporting 
 electrolyte , 0.1 m tbapf6 in mecn ; sweep rate , 100 mv / s .
quartz crystal microbalance ( qcm ) 
 measurements were used to determine 
 the surface coverage of the antibodies ( figure 2c ) .
the functionalized qcm crystal was placed in phosphate - buffered 
 saline solution ( pbs , ph 7.2 ) , and the frequency was allowed to stabilize 
 before the addition of a solution containing anti - dnp igg antibodies 
 ( figure 2c ) .
acoustic impedance methods ( supporting information figure s2 ) confirmed that 
 the films were rigid , and the sauerbrey equation was used to relate 
 the change in frequency to the mass of immobilized antibodies .
the 
 poegma brushes were tested for nonspecific adsorption in control experiments 
 by incubating the qcm crystals modified with poegma - dnp in a solution 
 of nonspecific antibodies .
no significant change in the frequency 
 was observed ( supporting information figure 
 s2 ) , confirming that nonspecific antibodies do not bind to the brushes . 

furthermore , no significant increase in the amount of h2o2 was observed under these control conditions ( vide infra ) 
 ( supporting information figure s3 ) .
two types of platforms were used , one on silicon and another on 
 a qcm crystal , in both cases presenting the polymer brushes adjacent 
 to the photosensitizer on the same surface ( figure 3a and b ) .
our ultimate device is designed to employ silicon 
 chips . however , for calibration purposes , qcm crystals were used to 
 quantify the mass of the bound antibodies . for the silicon chips , 

gold was evaporated onto 1  2 cm silicon wafer pieces 
 in a grid pattern .
the photosensitizer was electropolymerized on the 
 gold ( vide infra ) , whereas the polymer brushes , grown as depicted 
 in figure 2a , were confined to the silicon 
 oxide squares ( figure 3a ) .
the same grid pattern 
 was used for a qcm crystal ( figure 3b ) . however , 
 because both the polymer brushes and photosensitizer were polymerized 
 from a gold surface on the qcm crystal , a series of steps was employed 
 to prevent the two films from overlapping
. first , a photoresist was 
 spin coated onto the qcm crystals , exposed and developed to pattern 
 the grid .
next , the thiol functionalized atrp initiator was immobilized 
 on the exposed gold surface , and the photoresist was subsequently 
 removed .
finally , the photosensitizer was electropolymerized on the 
 sections where the photoresist was removed , and polymer brushes were 
 then grown from the regions containing the immobilized atrp initiator . 

this specific order of patterning and immobilization of the different 
 components allowed for the maximum yield of both photosensitizer and 
 polymer brushes on the same surface .
as described above , the 
 acwop process requires singlet oxygen , o2 * , 
 which can be generated from ambient oxygen o2 through the use of a photosensitizer , such as 
 [ ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] ( [ ru(v - bpy)3 ] ) .
we found that electropolymerized 
 films of [ ru(v - bpy)3 ] ( figure 3c ) on the gold electrode immediately 
 adjacent to the brushes maximized production of h2o2 .
typical coverage of photosensitizer was 1.5(0.5 )  
 10 mol / cm , corresponding to a thickness 
 of ca .
in addition to enhancing the signal - to - noise ratio , 
 our results showed a significant increase in the acwop h2o2 signal for the adjacent , immobilized photosensitizer 
 compared to photosensitizer in solution .
this increase is likely the 
 result of having the singlet oxygen generated in close proximity to 
 the antibody .
corresponding to a mean square distance 
 ( msd ) diffusion of less than 0.5 m , assuming a diffusion coefficient 
 of 2  10 cm / s . generating the singlet oxygen in close proximity to the 
 antibody clearly enhances the sensitivity of the biosensor . during 
 the course of developing this biosensor we tested a number of other 
 photosensitizers , including those used previously in acwop experiments , and we found these to be suboptimal . while there 
 may be more efficient singlet oxygen sensitizers in solution , in our 
 view [ ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] represents an almost ideal sensitizer in that it can 
 be readily polymerized , giving rise to the above - mentioned proximity 
 advantage , and the resulting films are conformal and quite robust . 

moreover , the complex is easily synthesized and 
 purified , and solutions can be used multiple times . to generate o2 * , and consequently h2o2 , the qcm crystal containing the immobilized 
 antibodies and electropolymerized sensitizer was immersed in 3 ml 
 of pbs ( ph 7.2 ) and exposed to uv irradiation for 60 min .
we found 
 that 60 min with a broad wavelength source was optimal , as longer 
 exposure times resulted in a decrease in signal and increase in background , 
 possibly due to uv damage of the antibody ( supporting 
 information figure s4 ) .
the h2o2 generated was quantified using square - wave 
 voltammetry ( swv ) ( figure 4 ) , which provides 
 high sensitivity at low analyte concentrations .
amplex red ( n - acetyl-3,7-dihydrophenoxazine ) 
 reacts with h2o2 in a 1:1 ratio in the presence 
 of horseradish peroxidase ( hrp ) to produce resorufin ( 7-hydroxy-3h - phenoxazin-3-one ) , which is fluorescent and also exhibits 
 a reversible redox response .
the lowest 
 concentration of h2o2 detected with swv was 
 0.33 nm ( figure 4a , b ) .
( a ) square wave voltammetry 
 of 10 m amplex red and 0.2 u / ml 
 horseradish peroxidase ( hrp ) with varying concentrations of h2o2 to construct a ( b ) calibration curve . supporting 
 electrolyte was pbs , ph 6.0 .
step size , 5 mv ; amplitude , 25 mv ; frequency , 
 25 hz .
( c ) square wave voltammograms showing detection of h2o2 via reduction of resorufin at a 3 mm glassy carbon 
 working electrode after irradiation of the immunosensors surface 
 with uv light .
black short dashed line is 10 m amplex red ; 
 red long dashed line is 10 m amplex red with 0.2 u / ml hrp in 
 the absence of antibody ; blue solid line is 10 m amplex red 
 with 0.2 u / ml hrp in the presence of adsorbed antibody .
step size , 
 5 mv ; amplitude , 25 mv ; frequency , 25 hz . a 1.5 ml aliquot of each 
 irradiated solution in pbs , ph 7.2 , was diluted to 5.0 ml using pbs , 
 ph 6.0 , and then amplex red and hrp solutions were added after deaerating 
 the pbs .
square wave voltammetry was subsequently carried out . assuming a surface coverage of 
 antibodies of 5  10 mol / cm , 
 and a liquid thickness of 100 m ( a value 
 readily achievable in a microfluidic platform ) , a single turnover 
 per immobilized antibody would give rise to a peroxide concentration 
 of 500 nm , clearly well above our detection limit ( less than 0.5 nm ) . 
 in comparative measurements we confirmed that swv detection is much 
 more sensitive than the fluorescence assay ( supporting 
 information figure s5 ) . to quantify the number of mole 
 equivalents of h2o2 generated per antibody
, 
 we evaluated h2o2 produced in the presence and 
 absence of the immobilized antibody 
 ( figure 4c ) .
an aliquot of the irradiated solution 
 was diluted with ph 6.0 pbs , followed by addition of amplex red and 
 hrp .
swv was used to measure the amount of resorufin , which reduces 
 near 0.2 v versus ag / agcl , at a glassy carbon electrode . a 
 readily quantifiable increase in reduction
current ( i.e. , the amount 
 of h2o2 produced ) resulted from the presence 
 of adsorbed antibody . using a calibration curve ,
( figure 4a , b ) . the ratio of h2o2 per 
 antibody was 6401200 , which substantially exceeds the previously 
 reported values .
although this difference 
 may be due , in part , to an increase in temperature , the biggest influence 
 may be the generation of o2 * in close proximity 
 to the antibody ( vide supra ) , such that this highly 
 reactive species is more readily intercepted by the antibody to generate 
 h2o2 . for any particular application , further 
 calibration with reference antibody stocks will allow the concentration 
 of antibodies in the test sample to be determined .
in summary , we have developed a general immunobiosensor platform , 
 employing patterned polymer brushes with photosensitizer films , based 
 on the electrochemical detection of h2o2 at 
 clinically relevant concentrations generated through the acwop .
we 
 have shown that h2o2 at concentrations as low 
 as 0.33 nm can be measured in a biosensor device .
antibodies at a 
 surface coverage of 5  10 mol / cm generate more than 25  10 mol h2o2/cm ( or > 250 m h2o2 , assuming a volume of 1 cm  1 cm  100 m ) 
 in 60 min .
however , h2o2 can be readily quantified 
 well below this concentration ( see figure 4a ) , and our tests indicate that this device can detect less than 
 3 pg antibodies in a 10 l sample ( 2 pm ) .
this compares quite 
 favorably to the most sensitive elisas , which require secondary agents 
 and additional procedural steps . as for the elisa ,
a challenge is 
 to ensure sufficiently high signal - to - noise , which can be limited 
 by nonspecific antibody binding .
the poegma brushes in our platform 
 appear to excel in this regard , and we will continue to develop these 
 as we test samples including high concentrations of nonspecific antibodies , 
 resembling typical blood serums . eliminating the use of secondary 
 antibodies , as in the case of elisa ,
reduces the chances for nonspecific 
  false positives  and furthermore makes our platform 
 appealing for low cost applications .
moreover , since the acwop is 
 a general characteristic of antibodies , our approach can , in principle , 
 be applied to antibodies of virtually any specificity , class , or species . 

we found results obtained from antibodies adsorbed on the platform 
 on a silicon chip to be similar to those obtained using the qcm crystal 
 ( supporting information figure s6 ) .
we 
 are currently exploring the incorporation of this approach into a 
 microfluidic platform , which would expedite wash steps to minimize 
 nonspecific binding , as well as delivery of the final test solution 
 for swv measurement of h2o2 concentration in 
 a separate part of the device . in the long run
this device may also 
 be useful for quantitatively assessing affinities of specific antibodies 
 in a way not possible with elisas because of their need for secondary 
 antibodies that complicate the binding analysis .
such a microfluidic 
 platform , in combination with flexible electronics ( again recall that 
 [ ru(4-vinyl-4-methyl-2,2-bipyridine)3 ] can be electropolymerized as conformal films ) should eventually 
 facilitate widespread use and field deployment .
 infectious diseases , 
 such as influenza , present a prominent global 
 problem including the constant threat of pandemics that initiate in 
 avian or other species and then pass to humans . 
 we report a new sensor 
 that can be specifically functionalized to detect antibodies associated 
 with a wide range of infectious diseases in multiple species . 
 this 
 biosensor is based on electrochemical detection of hydrogen peroxide 
 generated through the intrinsic catalytic activity of all antibodies : 
 the antibody catalyzed water oxidation pathway ( acwop ) . 
 our platform 
 includes a polymer brush - modified surface where specific antibodies 
 bind to conjugated haptens with high affinity and specificity . 
 hydrogen 
 peroxide provides an electrochemical signal that is mediated by resorufin / amplex 
 red . 
 we characterize the biosensor platform , using model anti - dnp 
 antibodies , with the ultimate goal of designing a versatile device 
 that is inexpensive , portable , reliable , and fast . 
 we demonstrate 
 detection of antibodies at concentrations that fall well within clinically 
 relevant levels . 
Results and Discussion
Conclusion