there is considerable 
 academic and commercial interest in the design 
 of biocompatible nonfouling surfaces for biological fluids , particularly 
 blood plasma and serum .
protein fouling can damage biomedical 
 devices , including coronary stents , ear 
 grommets , and guide wires ; it may also compromise the performance of biosensors , induce opsonization , and trigger clearance of 
 nanocarriers from the bloodstream .
adsorbed 
 proteins increase the bioburden for both external and intraocular contact lenses , 
 and play a critical role in thrombus formation in cardiovascular implants .
nonfouling surfaces are important in biosensors , 
 and in fundamental studies of biomaterial interfaces , because for 
 recognition - based binding of analytes , it is first necessary to eliminate 
 nonspecific binding . in array - based biosensors ,
nonfouling surfaces 
 must be capable of modification to yield spatially selective binding 
 of probe molecules .
poly(ethylene glycol ) ( peg)-based surface 
 coatings are known to 
 offer high resistance toward nonspecific protein adsorption and cell 
 adhesion .
however , peg - based materials 
 can be oxidized under physiological conditions , which can lead to activation of complement responses . over the past decade or so
, much research activity 
 has been focused on the design of alternative nonfouling biomaterials , 
 which has led to considerable advances in the design of novel biocompatible 
 materials .
various new surface coatings , such as self - assembled monolayers 
 ( sams ) , grafted polymer layers , and polymer brushes , have been proposed that substantially reduce protein absorption .
polymer brushes are polymer chains that are tethered to either 
 a planar or a colloidal surface , usually via just one chain - end .
the 
 most effective strategy for the synthesis of well - defined polymer 
 brushes is the so - called  grafting from  approach .
many controlled / living polymerization techniques ( clp ) , including 
 living anionic / cationic polymerization , nitroxide - mediated polymerization 
 ( nmp ) , and reversible addition  fragmentation 
 chain transfer ( raft ) polymerization , have been utilized for the chain growth reaction . arguably the most popular formulation is based on atom 
 transfer radical polymerization ( atrp ) .
this approach 
 has been exploited to prepare a wide range of functional polymer brushes 
 from many types of surfaces , including gold , silica , mica , base metals , 
 etc .
poly[oligo(ethylene glycol ) 
 methacrylate ] ( poegma ) brushes exhibit 
 exceptional resistance to protein adsorption from blood serum , and 
 also cell adhesion .
for example , katira et al . found 
 that adsorption of kinesin onto a 50 nm poegma brush was 20 times 
 lower than that found for monohydroxy - capped ( triethylene glycol)-terminated 
 sam surfaces .
particular attention has been paid to stimulus - responsive polymer brushes based on either polyacids 
 or polybases .
these polyelectrolytic brushes are typically responsive 
 to changes 
 in ph or ionic strength . for example , the stimulus - responsive 
 behavior of poly(acrylic acid ) brushes has been studied by ayres et 
 al .
collapsed brushes were observed below 
 the pka of the brush , whereas highly anionic 
 swollen brushes were formed above this critical value .
the ph - responsive behavior of poly(methacrylic 
 acid ) brushes , which enable switching between collapsed and stretched 
 brush conformations at different ph , has been investigated using ellipsometry 
 and atomic force microscopy ( afm ) . complementary 
 behavior has been reported for various weak polybase brushes based 
 on either poly(2-(diethylamino)ethyl methacrylate ) ( pdeaema ) , or poly(2-(dimethylamino)ethyl methacrylate ) 
 ( pdma ) .
polymers containing zwitterionic structural 
 units have been used 
 for a wide range of biomedical and engineering applications .
such 
 surface coatings are highly resistant to nonspecific protein adsorption , 
 bacterial adhesion , and biofilm formation .
jiang 
 and co - workers have demonstrated that glass slides grafted with two 
 zwitterionic polymers , poly(sulfobetaine methacrylate ) ( psbma ) and 
 poly(carboxybetaine methacrylate ) ( pcbma ) , exhibit exceptional resistance 
 to fouling . in some cases the zwitterionic 
 character is insensitive to the solution ph ( e.g. , poly(sulfobetaine 
 methacrylate ) ( psbma ) , and poly(2-(methacryloyloxy)ethyl 
 phosphorylcholine ) ( pmpc ) ) , whereas in 
 other cases ph - sensitivity can be observed .
for example , zwitterionic 
 poly(carboxybetaine methacrylate ) brushes exhibit zwitterionic character 
 at neutral ph , but cationic polyelectrolytic character at low ph .
as far as we are aware , there has been 
 relatively little work focused 
 on polymers containing amino acid motifs as side - chains .
for example , rosen et al . reported that cysteine - functionalized 
 silica nanoparticles resisted protein fouling when challenged with 
 either lysozyme or bovine serum albumin ( bsa ) .
azzaroni and co - workers grew cationic poly(methacryloyl - l - lysine ) brushes from mesoporous silica using surface - initiated 
 radical polymerization with the aim of modulating ionic transport 
 via ph variation .
liu et al . prepared 
 zwitterionic poly(serine methacrylate ) ( pserma ) brushes on a planar 
 gold substrate using surface - initiated photoiniferter - mediated polymerization 
 ( si - pimp ) for evaluation as a potential antibiofouling material .
very recently , jiang and co - workers have reported 
 the synthesis of new polycarboxybetaine brushes based on two new amino - acid - based 
 methacrylic monomers prepared via three - step and five - step protocols , 
 respectively . herein a new zwitterionic 
 monomer , cysteine methacrylate ( cysma ) , 
 has been conveniently synthesized via thia - michael addition of cysteine 
 to a commercial methacrylate - acrylate precursor ( see scheme 1a ) .
this facile synthesis is particularly noteworthy 
 because it is conducted on a multigram scale in aqueous solution 
 without recourse to protecting group chemistry .
this offers 
 the promise of greatly reduced cost compared to other zwitterionic 
 polymer brush systems such as pmpc .
si - atrp was then used to grow 
 pcysma brushes from planar silicon wafers ( see scheme 1b ) , and their stimulus - responsive behavior with respect to 
 changes in ph and ionic strength was investigated using ellipsometry , 
 atomic force microscopy ( afm ) , and surface zeta potential measurements . 

micro- and nanopatterned pcysma brushes were prepared via uv irradiation 
 using a photomask and interference lithography ( il ) , respectively . 

selective adsorption of fluorescent proteins on these model surfaces 
 was characterized by confocal fluorescence microscopy .
the long - term 
 aqueous chemical stability of pcysma brushes immersed in alkaline 
 media and the uv - induced photodegradation of dry pcysma brushes was 
 examined using x - ray photoelectron spectroscopy ( xps ) and afm .
finally , 
 complement depletion assays were used to compare the antibiofouling 
 performance of pcysma brushes to other well - known biocompatible pmpc 
 and poegma brushes .
cysteine methacrylate 
 ( cysma ) monomer was synthesized at 20 c in aqueous solution 
 by a selective thia - michael addition . a natural amino acid , l - cysteine ,
was reacted with 3-(acryloyloxy)-2-hydroxypropyl 
 methacrylate in the presence of a nucleophile catalyst ( dmpp ) , see 
 scheme 1a . thia -
michael addition proved to 
 be relatively efficient , with an overall yield of 94% being achieved 
 within 2 h. the chemical structure of the purified monomer was confirmed 
 by h and c nmr spectroscopy , mass spectroscopy 
 and elemental microanalyses ( see supporting information ) .
films 
 formed by the adsorption of 3-aminopropyl triethoxysilane ( aptes ) 
 onto silica were reacted with 2-bromoisobutyryl bromide ( bibb ) to 
 generate 3-(2-bromoisobutyramido)propyl triethoxysilane ( bibb - aptes ) .
the expected chemical composition of bibb - aptes 
 was confirmed by xps studies ( c 1s , br 3d and n 1s core - line spectra ) , 
 and the surface zeta potential of bibb - aptes film was measured at 
 different ph solution ( figures s2 and s3 , see supporting information ) .
pcysma brushes were grown from 
 bibb - aptes - functionalized silicon wafers via si - atrp , which is known 
 to be an effective and convenient technique for the synthesis of dense 
 uniform polymer brushes with controlled thickness ( see scheme 1b ) .
the cu(i)/cu(ii ) molar ratio , solvent type , 
 and cysma / catalyst molar ratio in the aqueous atrp formulation were 
 varied so as to optimize the brush growth conditions .
figure 1 shows the evolution of pcysma brush thickness over 
 time at 20 c using [ cysma]/[cu(i)br ] molar ratios of 30 , 100 , 
 and 150 .
ellipsometric dry brush thicknesses of up to 27  3 
 nm were achieved after 270 min .
120 min , at which point there was a reduction in 
 the rate of growth .
higher [ cysma]/[cu(i)br ] molar ratios led to thicker 
 pcysma brushes , as expected .
evolution in ellipsometric dry brush thickness 
 with polymerization 
 time for the synthesis of pcysma brushes via surface - initiated atrp 
 in deionized water at 20 c .
conditions : ( red squares ) [ cysma]:[cubr]:[cubr2]:[bipy ] molar ratio = 30:1.0:0.5:3 ; ( blue circles ) [ cysma]:[cubr]:[cubr2]:[bipy ] molar ratio = 100:1.0:0.5:3 ; ( green diamonds ) [ cysma]:[cubr]:[cubr2]:[bipy ] molar ratio = 150:1.0:0.5:3 .
high resolution c 1s , n 1s , and s 2p xps spectra were acquired 
 for pcysma dry brushes in order to confirm their chemical structure 
 ( see figure 2 ) .
xps c 1s spectra were fitted 
 using three components with binding energies of 285.0 , 286.3 , and 
 288.7 ev , which correspond to c  c , c  o / c  n , and o = c  o , respectively .
the c  c , c  o / c  n , and o = c  o 
 atomic ratios calculated from the fitted c 1s spectrum were 2:1.5:1 , 
 which is close to the theoretical ratio of 2:1.3:1 . the binding energy 
 of the o = c  o component was reduced 
 by ca .
0.6 ev compared to the expected value for a carboxylic acid , 
 consistent with previous reports for anionic carboxylate carbon atoms .
the n 1s core - line spectrum recorded for the 
 pcysma brush ( dried at ph 6 ) was fitted using two components centered 
 at 399.0 and 401.5 ev , which correspond to c  nh2 and c  nh3 species .
it is estimated that about 
 80% of the surface primary amine groups are protonated .
the high resolution 
 s 2p spectrum was fitted using two s 2p3/2 and s 2p1/2 components with binding energies of 163.5 and 164.5 ev , 
 respectively .
the relative intensities of these components is approximately 
 2:1 , as expected for 2j + 1 spin 
tapping mode afm topographical images 
 were acquired for pcysma 
 brushes both in the dry state and also immersed in pbs ( see figure 
 s4 , supporting information ) .
the root - mean - square 
 ( rms ) surface roughness averaged across the entire image area was 
 0.5  0.2 nm in the dry state . however , the pcysma brush became 
 somewhat smoother when immersed in pbs solution , with an rms roughness 
 of approximately 0.3  0.1 nm .
the carboxylic 
 acid and amine groups in each cysma residue confer ph - responsive behavior 
 on the pcysma brushes . in this sense , these weak polyacid / weak polybase 
 brushes differ significantly from permanently zwitterionic betaine - based 
 brushes such as pmpc or psbma , or indeed pcbma ( which only possesses 
 weak polyacid character ) . this complex ph - sensitive behavior was studied 
 using both ellipsometry and afm .
high resolution x - ray photoelectron spectra 
 recorded for a pcysma 
 brush of 12 nm dry thickness : ( a ) c 1s spectrum , ( b ) n 1s spectrum , 
 ( c ) s 2p spectrum .
the ph - modulated dimensions 
 of a pcysma brush ( dry brush thickness 
 = 15 nm ) immersed in an aqueous solution were investigated by varying 
 the solution ph over a wide range . to enable the brush thickness to 
 be measured by atomic force microscopy , patterned samples were prepared 
 by uv irradiation of bibb - aptes films through a mask . in exposed regions 

the initiator remains intact in the masked areas , from which 
 brushes may be grown subsequently by atrp .
the br 3d signal in the xps spectrum was found to decrease in intensity 
 with uv exposure and became undetectable after a dose of 2.2 j cm . in the present study
, bibb - aptes films were exposed 
 to the same dose using a 2000 mesh electron microscopy grid as a photomask 
 to generate a micropatterned bibb - aptes monolayer , which was subsequently 
 utilized to grow micropatterned pcysma brushes by atrp .
the initial 
 micropatterned substrate did not exhibit any height contrast between 
 the uv - exposed and unexposed areas .
figure 3 shows an afm topographical image acquired after atrp had been carried 
 out .
significant height differences were observed between irradiated 
 areas ( squares ) and nonirradiated areas ( bars ) , as expected ( figure 3 ) .
tapping - mode afm of the periodic brush 
 height recorded for a micropatterned 
 pcysma brush : topographical image ( left ) , cross section analysis ( right ) . 

image size : 75  75 m , z - range 
 050 nm . the ellipsometric data 
 ( see figure 4a ) indicate 
 that the pcysma brushes become protonated at a ph less than ca . 2.0 
 and
below 
 its pka , the carboxylic acid group is 
 no longer ionized , while all the amine groups remain protonated .
thus 
 the brush layer stretches away from the surface because of the strong 
 lateral electrostatic repulsion between adjacent cationic chains . 

in contrast , the amine groups become deprotonated above their pka while the carboxylic acid groups remain ionized ; 
 hence , the brushes acquire anionic character and hence become highly 
 swollen above ph 9.8 .
the mean brush thickness increased from 15 nm 
 ( dry state ) to approximately 36 nm at ph 1.5 .
between ph 1.6 and 2.0 , 
 there is a gradual reduction in brush thickness from 36 to 28 nm because 
 of the higher degree of ionization of the carboxylic acid groups . 

the mean pcysma brush thickness then remains constant at approximately 
 26 nm between ph 2.0 and ph 9.5 ; this corresponds to the zwitterionic 
 brush regime .
finally , the brush thickness increases from 28 nm to 
 approximately 3435 nm between ph 9.5 and ph 9.8 as the amine 
 groups become deprotonated and the brush chains acquire anionic character . 
 in summary , the pcysma brushes exhibit zwitterionic character at intermediate 
 ph values ( i.e. , pka < 
 ph < pka2 ) 
 and are somewhat less swollen than their polyelectrolytic forms under 
 these conditions . variation in the in situ brush thickness 
 with 
 ph for a pcysma brush of 15 nm dry thickness immersed in aqueous solution 
 by ( a ) ellipsometry and ( b ) afm .
( c ) surface zeta potential vs ph 
 curves obtained for pcysma brushes using a malvern dip cell .
pcysma 
 brush ( original dry brush thickness = 22 nm ) immersed in aqueous solutions 
 of varying ph in the presence of 1 mm kcl as background electrolyte .
measurements were made by afm 
 on a micropatterned pcysma brush , 
 similar to the one shown in figure 3 , immersed 
 in aqueous solutions of varying ph .
the height difference was measured 
 between the brush - functionalized bars and the square regions from 
 which the initiator was removed by uv exposure .
the 
 brush height increases from about 15 nm in the dry state to 44 nm 
 when immersed in an acidic solution ( ph < 1.5 ) .
the brush height 
 is gradually reduced between ph 1.6 and 2.0 , and then remains constant 
 at approximately 28 nm between ph 2.0 and 9.5 .
finally , there is a 
 gradual increase in brush height between ph 9.5 and 9.7 , and a maximum 
 brush thickness of 40 nm is observed above ph 10 .
the pka of the carboxylic acid groups in the pcysma brush is 
 estimated to be approximately 1.7 , compared to a pka of 2.0 for the carboxylic acid group of l - cysteine . 

similarly , the amine pka of the pcysma 
 brushes is estimated to be 9.6 , whereas it is reported to be 10.2 
 for l - cysteine .
these differences 
 are attributed to the well - known polyelectrolyte effect : the ph response 
 of polyacids and polybases is always weaker than the corresponding 
 small molecules , since polymer chains tend to resist the build - up 
 of local charge density .
these 
 afm observations are in generally good agreement with the 
 ellipsometric data , with minor differences most likely arising because 
 of uncertainties in the refractive index of 1.50 estimated for the 
 pcysma brush when modeling the ellipsometric data ( n.b . this parameter 
 is also likely to vary with the solution ph ) .
there has recently 
 been interest in the use of ph - responsive polymers 
 for various applications , for example for gating channels or , more recently , for the reversible trapping 
 and release of nanoparticles or proteins .
pcysma is an attractive material for such applications because of 
 the facile monomer synthesis and the unusual ph - responsive properties 
 of the polymer .
the determination of surface zeta potentials 
 for planar surfaces 
 has been recently described using a new commercial dip cell developed 
 by malvern instruments . however , as far 
 as we are aware , this technique has only recently applied to characterize 
 ph - responsive polymer brushes .
surface 
 zeta potentials were determined for pcysma brushes over a wide range 
 of solution ph , see figure 4c .
positive surface 
 zeta potentials of + 15  5 mv were observed below ph 2 for protonated 
 pcysma brushes .
between ph 2.5 and 9.0 , the pcysma brush exhibited 
 weakly negative surface zeta potentials ( 5 to 10 mv ) . 
 in principle
, the brush layer should be in its zwitterionic form over 
 this ph range , so zero surface zeta potentials might be expected . 

the observed weakly anionic character may indicate preferential adsorption 
 of cl and/or oh from the bulk 
 solution . above ph 9 , the surface zeta potential becomes much more 
 negative ( 40  8 mv ) , indicating deprotonation of the 
 amine groups within the pcysma brush .
overall , these surface zeta 
 potential data are consistent with the changes in brush thickness 
 observed using ellipsometry and afm discussed above and support our 
 interpretation of the complex ph - modulated behavior exhibited by this 
 doubly ph - responsive brush in aqueous solution .
pcysma brushes 
 exhibited good chemical stability in either acidic 
 or neutral aqueous solution .
ellipsometric studies indicate a gradual reduction 
 in brush thickness from 10 to 7 nm at ph 9 ( see figure s5 , supporting information ) . at ph 10
, the mean brush 
 thickness is reduced from 10 to 3 nm within 48 h. finally , a brush 
 thickness of just 1.5 nm is observed within 24 h at ph 11 .
in contrast , 
 no discernible change in the ellipsometric brush thickness is observed 
 after 9 days at ph 8 .
thus it seems that , although these brushes are 
 susceptible to base - catalyzed hydrolysis , they nevertheless possess 
 sufficient stability under physiological conditions to be considered 
 for biomedical applications .
, a primary amine group could react intramolecularly 
 with an ester carbonyl that is located either on the same cysma residue 
 or on an adjacent repeat unit to form an amide .
if the primary amine 
 group reacts with an ester carbonyl on a neighboring pcysma chain , 
 a cross - link can be formed . at higher ph , base - catalyzed ester hydrolysis 
 is also likely to occur .
no changes are observed in the x - ray 
 photoelectron spectra recorded 
 for pcysma brushes after their immersion in an aqueous solution at 
 ph 8 for 48 h. however , exposing pcysma brushes to alkaline media 
 ( ph > 9 ) led to various spectroscopic changes over this time scale 
 ( figure s6 , see supporting information ) . 

a significant reduction in the c 1s intensity is observed , which indicates 
 substantial mass loss and implies chemical degradation .
the s 2p and 
 n 1s signals each become attenuated over time , indicating loss of 
 the cysteine fragment from the brush layer .
however , it is emphasized 
 that there is no evidence for chemical degradation at neutral ph , 
 which suggests that this new zwitterionic pcysma brush may offer potential 
 applications as a surface coating under physiological conditions .
the effect of ionic strength 
 on the pcyma brush dimensions was also investigated by adding salt 
 ( nacl ) . according to the literature
figure 5 shows the change in pcysma 
 brush thickness in the presence of added salt at ph 6 , as judged by 
 ellipsometry .
the mean brush thickness increased monotonically from 
 28.0 to 30.5 nm when immersed in aqueous solutions containing 0.3 
 to 1.5 m nacl .
the salt screens the interchain attractive electrostatic interactions , which allows modest brush expansion . 

in contrast , the addition of salt to polyelectrolytes screens the repulsive electrostatic interactions between adjacent chains , 
 leading to their collapse ( see figure 5 ) .
thus , 
 at ph 1.5 , there is a gradual reduction in the wet cationic brush thickness from 43 to 35 nm on addition of salt .
similar behavior 
 is observed when salt is added to anionic pcysma 
 brushes at ph 9.8 : the ellipsometric wet brush thickness is reduced 
 from 48 to 35 nm in the presence of 1 m nacl . in situ ellipsometric wet thickness of pcysma 
 brushes as a function of added salt at 20 c .
red squares : immersed 
 in aqueous solution at ph 6 , original dry thickness 15 nm .
blue circles : 
 immersed in aqueous solution at ph 1.5 , original dry thickness 18 
 nm .
green diamonds : immersed in aqueous solution at ph 9.8 , original 
 dry thickness 18 nm .
pcysma 
 brushes grown from silicon wafers were subjected to various 
 uv doses at 244 nm .
significant changes were observed in the c 1s 
 spectra during this irradiation ( figure 6a ) . 
 in particular , there is a systematic reduction in the c 1s signal 
 intensity with longer exposure times ( 5 min ) , which suggests that 
 the brush thickness is reduced with increasing uv exposure .
moreover , 
 a new component is observed at 288 ev which is assigned to 
 the formation of surface aldehyde groups .
( a ) peak - fitted c 1s 
 spectrum of pcysma brush after uv irradiation 
 ( total dose 10.4 j cm ) including the aldehyde 
 component at 288 ev .
( b ) c 1s and ( inset ) f 1s peak - fitted x - ray photoelectron 
 spectra recorded for pcysma brushes exposed to uv radiation ( total 
 dose = 48 j cm ) and subsequently immersed into 
 an ethanolic solution of trifluoroethylamine .
these surface aldehyde groups were reacted with a perfluorinated 
 primary amine to produce imine linkages via schiff base chemistry ; 
 this is a well - known assay that has the advantage of enhancing the 
 xps sensitivity .
figure 6b shows the c 1s and 
 f 1s spectra recorded for a pcysma brush after an exposure of 14 j 
 cm , followed by immersion in an ethanolic solution 
 of trifluoroethylamine . for the treated sample , the observation of 
 a strong f 1s peak at 688.5 ev and a new component at ca .
293 ev in 
 the c 1s region ( assigned to the cf3 unit 
 in the amide product ) provide good evidence that the reaction has 
 taken place .
a uniform pcysma brush - functionalized silicon wafer 
 was exposed 
 to uv radiation using a photomask and subsequently imaged using afm 
 ( figure 7 ) . after exposure , the exposed regions 
 ( squares ) exhibited reduced height compared to the masked regions 
 ( bars ) . as the exposure increased the height difference between the 
 masked and exposed regions increased .
12 nm ; thus the mean 
 erosion rate was estimated to be 0.25 nm j cm .
( a ) difference in height between masked and exposed areas as a 
 function of dose for pcysma brushes samples exposed to uv radiation 
 (  = 244 nm ) through a mask ( original dry brush thickness = 
 18 nm ) .
images b  d show 75  75 m afm 
 topographical images ( with a common vertical range of 20 nm ) as a 
 function of uv dose .
nanostructures were fabricated by photodegradation of a pcysma 
 brush using a dual - beam interferometer .
a coherent uv laser beam was directed toward a mirror , and the pcysma 
 brush - coated silicon wafer was placed at an angular separation of 
 2. half of the laser beam fell on the mirror , from where it 
 was reflected onto the sample to interfere with the other half of 
 the beam , forming a pattern with a sinusoidal variation in intensity 
 and a period of /2sin  , where  is the laser wavelength 
 ( 244 nm ) .
the pcysma brush was 
 exposed to a uv dose of 13.2 j cm at angles of 
 10 , 20 , 30 , and 45 , yielding pcysma brush 
 nanolines with mean line widths ( full width at half - maximum , fwhm ) 
 of 350  5 nm , 245  5 nm , 130  5 nm , and 100  
 5 nm , respectively , and heights of 3  1 nm , 3  0.5 nm , 
 3  0.5 nm , and 2  0.5 nm , with periods of 730  20 
 nm , 455  15 nm , 235  15 nm , and 190  10 nm , respectively .
pcysma brushes exhibit excellent 
 resistance toward protein adsorption , but this protein resistance 
 was found to be lost after exposure to uv light .
they were then immersed 
 in an aqueous solution containing 10 g ml gfp in pbs ( see figure 8a ) . in the masked 
 areas ( bars in figure 8a )
no fluorescence can 
 be detected within the limits of sensitivity , which indicates that 
 the pristine pcysma brush strongly resists protein adsorption . in 
 contrast
, strong green fluorescence is observed from the uv - irradiated 
 square regions , indicating efficient selective gfp immobilization 
 within these areas .
such a well - defined gfp pattern within a pcysma 
 brush ( combined with minimal background fluorescence ) suggests that 
 this new photopatternable antibiofouling brush layer offers considerable 
 potential for various biomedical applications .
control samples that 
 had not been exposed to uv light ( supporting information , figure s8 ) exhibited no fluorescence .
confocal fluorescence 
 images obtained for pcysma brushes subjected 
 to a green fluorescent protein ( gfp ) challenge : ( a ) gfp protein is 
 immobilized within bright green squares after selective degradation 
 of pcysma using a photomask ( dark areas represent the unexposed pcysma 
 brush ) ; ( b ) green fluorescent protein ( gfp ) immobilized in nanopatterned 
 lines after selective degradation of pcysma using a two - beam interferometer 
 at  = 10. protein nanopatterns were fabricated by interferometric lithography 
 ( il ) ( figure 11b ) . a pcysma brush ( mean dry 
 brush thickness = 15 nm ) was exposed to a uv dose of 13.2 j cm at an angle of 10 , and immersed in a 10 g 
 ml solution of gfp in pbs .
a relatively small 
 angle was selected , leading to a comparatively large period and fwhm , 
 to enable characterization of the structures by confocal microscopy , 
 which is a diffraction - limited technique .
the green lines indicate 
 that gfp has adsorbed to the chemically modified brush chains , corresponding 
 to regions that were exposed to maxima in the interferogram , whereas 
 the unmodified brush regions ( exposed to minima in the interferogram ) 
 continue to resist protein adsorption strongly .
individual lines are 
 well - resolved , and the mean line width was estimated to be 350 nm . for array - based biosensors , and for fundamental studies of interfacial 
 biological phenomena , it is important to be able to introduce protein - binding 
 regions into a protein - resistant surface .
the data shown in figure 8 exhibit high contrast between modified and unmodified 
 regions , indicating that very effective control of protein 
additionally , 
 the protein - resistant pcysma is converted to a protein - binding aldehyde 
 functionality in a single step , in contrast to the multistep processes 
 that are typically used to attach biomolecules to surfaces .
many studies 
 have assessed antibiofouling performance using single protein assays 
 [ i.e. , hsa , fibrinogen , and single complement proteins ( c3b ) ] . however , reduction of the absorption of a single 
 blood plasma protein is insufficient evidence for stealth - like properties . perhaps surprisingly , there are relatively few 
 literature reports that assess the antibiofouling performance of poly - zwitterion - based 
 surface coatings utilizing full blood plasma . in principle , antibiofouling assays that involve incubation with 
 whole human serum ( which contains a full set of complement proteins ) 
 and utilize cell membrane lysis as the end - point offer the potential 
 for mimicking possible activation through all the known complement 
 pathways .
the cytotoxicity of these 
 pcysma brushes was evaluated using a biological assay ( see supporting information ) .
there was no evidence 
 of cytotoxicity for human dermal fibroblasts exposed to pcysma brushes 
 over a wide range of brush thicknesses .
the mtt assay was used to 
 assess the extent of cell adhesion , which is a well - recognized requirement 
 for cell viability of adherent cells such as fibroblasts .
an adhesion study was performed for 24 h in 
 order to evaluate the antibiofouling performance of pcysma brushes 
 in conjunction with hdf cells . over this time period
, very few hdf 
 cells adhered to the pcysma brush - coated glass substrates ( see figure 9 ) .
optical microscopy studies suggested that very 
 few cells became attached to either the relatively short ( 47 
 nm ) brushes or those of intermediate thickness ( 1316 nm ) , 
 while no adhering cells at all were observed for the relatively thick 
 2428 nm brushes ( figure 9b ) .
when surface 
 attachment did occur , adhering cells exhibited a pseudospherical morphology 
 ( figure 9b ) , whereas the control cells were 
 both highly adherent and fully stretched .
this indicates that the 
 pcysma brushes strongly resist cell adhesion when immersed in culture 
 media in the presence of hdf cells over an extended incubation period .
pcysma brushes were grown 
 from identical 13 mm diameter circular glass slides to examine the 
 extent of cellular adhesion on these substrates .
both pcysma - coated and uncoated glass slides 
 were disinfected using ice - cold ethanol and placed at the bottom of 
 24-well plates .
hdfs cells were then added ( 1  10 cells per well ) , and cells were incubated for 24 h at 37 c . 

( a ) cell adhesion onto three series of pcysma brushes ( with dry brush 
 thicknesses of 47 nm , 1316 nm , or 2428 nm ) . 

n = 3 
 independent experiments were performed in duplicate wells . * * p < 0.01 .
( b ) optical micrographs obtained for hdf cells 
 growing on the brush surfaces after 24 h incubation .
a complement depletion assay was utilized to further 
 investigate 
 the effect of varying the pcysma brush thickness in the context of 
 protein fouling ( see figure 10 ) . on exposure 
 of the pcysma
brush - coated glass slides to human serum , all brushes 
 performed much better with regard to complement depletion than typical 
 non - adherent surfaces ( e.g. , non - tissue culture plastic and untreated 
 glass , p < 0.05 ) .
shorter and intermediate pcysma 
 brushes ( 47 nm and 1316 nm dry thicknesses , respectively ) 
 led to weak but detectable depletion compared to the zero depletion 
 control ( p < 0.05 ) . however , the relatively long 
 pcysma brushes ( 2428 nm dry thickness ) showed no statistically 
 significant differences compared to the zero depletion control ( see 
 figure 10 ) . these results confirmed the qualitative 
 observations reported in figure 9b , suggesting 
 that a certain minimum brush thickness is required for optimum antibiofouling 
 performance.figure 10complement depletion 
 assays performed on pcysma brushes of varying 
 thickness .
pcysma brushes were grown from 13 mm diameter circular 
 glass slides with mean dry brush thicknesses of 47 nm , 1316 
 nm , and 2428 nm and incubated with human serum ( 100 l ) 
 for 1 h at 37 c .
data were referenced 
 to water - lyzed erythrocytes ( 100% lysis control or zero depletion 
 control ) .
additional control surfaces were also used ( e.g. , non - tissue 
 culture ( tc ) plastic and an untreated glass slide ) .
n = 3 independent experiments were performed in duplicate wells . * p < 0.05 ( compared to the zero depletion control ) , + p < 0.05 ( compared to non - tc plastic ) ,  p < 0.05 ( compared to untreated glass ) .
pcysma brushes were grown from 13 mm diameter circular 
 glass slides with mean dry brush thicknesses of 47 nm , 1316 
 nm , and 2428 nm and incubated with human serum ( 100 l ) 
 for 1 h at 37 c .
data were referenced 
 to water - lyzed erythrocytes ( 100% lysis control or zero depletion 
 control ) .
additional control surfaces were also used ( e.g. , non - tissue 
 culture ( tc ) plastic and an untreated glass slide ) .
n = 3 independent experiments were performed in duplicate wells . * p < 0.05 ( compared to the zero depletion control ) , + p < 0.05 ( compared to non - tc plastic ) , 
p < 0.05 ( compared to untreated glass ) . finally , the complement depletion performance 
 of well - known antibiofouling 
 pmpc and poegma brushes was compared to pcysma brushes of comparable 
 dry thickness ( approximately 2530 nm in each case ) , see figure 11 . to maximize 
 the assay sensitivity ,
complement depletion was monitored 
 over a range of normal human serum ( nhs ) concentrations ( 100% , 50% , 
 and 25% ) , such that the zero depletion controls were still able to 
 completely lyze the cells ( see figure 11b ) . 

pcysma brushes proved to be slightly superior to the pmpc and poegma 
 brushes in their antibiofouling properties over the entire concentration 
 range ( figure 11a ) .
the former brushes exhibited 
 no discernible difference compared to the zero depletion control , 
 whereas the pmpc and poegma brushes each showed a small but significant 
 increase in complement depletion ( p < 0.05 ) .
( a ) pcysma , pmpc and poegma brushes of equivalent 
 thickness ( approximately 2530 nm ) were tested for complement 
 depletion and data were compared to both non - tissue culture plastic 
 control ( ntcp ) and untreated glass .
antibiofouling performance was 
 monitored over a range of human serum concentrations ( 100% , 50% , and 
 25% serum ) .
lower concentrations incurred errors because there were 
 insufficient complement proteins to lyze the cells , as shown in part 
 b. ( b ) n = 3 independent experiments were performed 
 in duplicate wells .
a 
 new amino acid methacrylate monomer , cysteine methacrylate ( cysma ) , 
 has been conveniently prepared on a 40-g scale without recourse to 
 protecting group chemistry via a facile high - yielding thia - michael 
 addition conducted in aqueous solution , offering the prospect of straightforward 
 and cost - effective scale - up .
this monomer was used to prepare novel 
 zwitterionic pcysma brushes via surface - initiated atom transfer radical 
 polymerization with mean brush thicknesses ranging between 4 and 28 
 nm .
for example , 
 the mean brush thickness increased significantly below ph 2 and above 
 ph 9.5 , as judged by ellipsometry and afm studies .
surface zeta potential 
 studies indicate that pcysma brushes acquire cationic character below 
 the former ph and anionic character above the latter ph , with zwitterionic 
 character being observed at intermediate ph .
modest brush swelling 
 occurs in the presence of added salt at ph 6 , which is attributed 
 to the well - known 
in contrast , conventional polyelectrolyte behavior , 
 i.e. , brush collapse , is observed on addition of salt at either low 
 ph or high ph , since the electrostatic repulsive forces between adjacent 
 charged chains are screened .
xps studies of photodegraded pcysma brushes 
 confirm that surface aldehyde groups are generated after prolonged 
 uv irradiation at 244 nm .
both micro- and nanostructured pcysma brushes 
 can be conjugated to either green fluorescent protein ( gfp ) via these 
 surface aldehyde groups .
pcysma brushes proved to be highly resistant 
 to adhesion when exposed to hdf cells , while complement consumption / depletion 
 assays confirm that such brushes exhibit superior antibiofouling performance 
 to that of well - known pmpc and poegma brushes .
finally , these pcysma 
 brushes exhibit good long - term stability under physiological conditions , 
 but are prone to base - catalyzed hydrolysis when immersed in mildly 
 alkaline solution ( ph > 8) .
 a new 
 cysteine - based methacrylic monomer ( cysma ) was conveniently 
 synthesized via selective thia - michael addition of a commercially 
 available methacrylate - acrylate precursor in aqueous solution without 
 recourse to protecting group chemistry . 
 poly(cysteine methacrylate ) 
 ( pcysma ) brushes were grown from the surface of silicon wafers by 
 atom - transfer radical polymerization . 
 brush thicknesses of ca . 
 27 
 nm were achieved within 270 min at 20 c . 
 each cysma residue 
 comprises a primary amine and a carboxylic acid . 
 surface zeta potential 
 and atomic force microscopy ( afm ) studies of the ph - responsive pcysma 
 brushes confirm that they are highly extended either below ph 2 or 
 above ph 9.5 , since they possess either cationic or anionic character , 
 respectively . at intermediate ph , 
 pcysma brushes are zwitterionic . 
 at physiological ph 
 , they exhibit excellent resistance to biofouling 
 and negligible cytotoxicity . 
 pcysma brushes undergo photodegradation : 
 afm topographical imaging indicates significant mass loss from the 
 brush layer , while xps studies confirm that exposure to uv radiation 
 produces surface aldehyde sites that can be subsequently derivatized 
 with amines . 
 uv exposure using a photomask yielded sharp , well - defined 
 micropatterned pcysma brushes functionalized with aldehyde groups 
 that enable conjugation to green fluorescent protein ( gfp ) . 
 nanopatterned 
 pcysma brushes were obtained using interference lithography , and confocal 
 microscopy again confirmed the selective conjugation of gfp . finally , 
 pcysma undergoes complex base - catalyzed degradation in alkaline solution , 
 leading to the elimination of several small molecules . 
 however , good 
 long - term chemical stability was observed when pcysma brushes were 
 immersed in aqueous solution at physiological ph . 
Introduction
Results and Discussion
Conclusions