the use of polymeric 
 materials in crystallization has a long history 
 of success in producing high - quality crystals of substances such as 
 inorganic and organic materials of low solubility . silica gels , for 
 example , are commonly used as polymeric materials in crystallization 
 experiments , although peo ( poly(ethylene 
 oxide ) ) and polyacrylamide have also been explored in the search of organic solvent 
 compatibility . in general , gels provide a convection - free environment , 
 while avoiding sedimentation of the growing crystals , and , in combination 
 with counterdiffusion setups , allow the search of a wide range 
 of supersaturation values in a single experiment . further experiments with other gels have 
 been conducted and explored 
 ( lorber et al . and refs herein ) as crystallization 
 media . agarose gels , for example , are very effective in reducing convection 
 even at low concentration values ( 0.12% w / v ) . these gels have also been found to be an effective filtering medium 
 to reduce the incorporation of impurities . it 
 has been shown that the use of agarose gels also improves crystal 
 quality , while facilitating protein crystal manipulation , 
 avoiding osmotic shock , and also serving 
 as cryo - protectant . these attractive 
 observations of increasing robustness of protein crystals occur mainly 
 due to the incorporation of the gel fibers into the crystal lattice . the aforementioned phenomenon is being explored to produce composite 
 protein  gel crystals with improved properties . similar observations have been recently obtained for various 
 inorganic crystals as well as gels of different nature . polymers such as poly(ethylene oxide ) and poly(vinyl alcohol ) , calcium alginate beads , or new synthetic polymers [ poly(n - isopropylacrylamide - co - n - butyl methacrylate ) ] and hydrophilic 
 polymer blocks [ poly(ethylene glycol ) ] have also been tested for the crystallization of biological macromolecules . gels may also serve as a buffer to control the supersaturation 
 rate within crystallization experiments . hughes and co - workers used 
 the counterdiffusion technique in a three - layer configuration 
 to produce large crystals of inorganic pyrophosphatase 
 ( ippase ) of millimeter size for neutron diffraction in capillaries 
 of up to 2.0 mm inner diameter . peg - based cross - linked hydrogels have been explored for many biological 
 as well as biomedical applications including sensors and controlled 
 drug delivery of macromolecules mainly due to their nontoxicity and non - immunogenicity . they are also known to protect peptides and 
 proteins against enzyme degradation by the formation of conjugates . to date , peg linear polymers have been employed mainly as crystallization 
 agents in solution . they could also be a very effective medium to 
 crystallize macromolecules due to their rigid structure and 
 their capacity to reduce protein solubility . however , contrary to 
 linear peg polymers , peg cross - linked hydrogels can be tailored to 
 have a specific pore size as well as to incorporate other desired 
 chemical moieties to modify the charge , etc . therefore , the main aim 
 of this work was to explore the feasibility of using cross - linked 
 peg - based hydrogels as crystallization media to grow protein crystals . two types of studies were conducted : one focused on the investigation 
 of the influence of the peg hydrogel on the resulting size and morphology 
 of the lysozyme - grown crystals in capillary counterdiffusion 
 experiments , and the second focused on the investigation of the effect 
 of pore size of the gel network on the nucleation and growth of lysozyme 
 crystals in counterdiffusion experiments . the outcome of these 
 experiments demonstrated that it is possible to obtain protein crystals 
 with incorporated peg hydrogel fibers . on the other hand , we demonstrated 
 that the diffusion of the crystallization agent could be controlled , 
 which may be exploited to obtain crystals of larger size for neutron 
 diffraction . poly(ethylene glycol ) 
 ( peg)-based hydrogels were prepared by mixing poly(ethylene glycol ) 
 monomethyl ether monomethacrylate ( pegma ) ( polysciences , warrington , 
 pa ) with a molecular weight of 1100 da ( manufacturer reports the molecular 
 weight of the product as 1000 , which represents the average molecular 
 weight of the peg chain only , thus the 1100 includes the molecular 
 weight of the methacrylate group ) with the cross - linker poly(ethylene 
 glycol ) dimethacrylate ( pegdma ) ( polysciences , warrington , 
 pa ) ( mw 1200 da ) . the monomer with the highest molecular weight was 
 selected in order to have the longest tethered chains that could potentially 
 interact with the protein . this monomer combination was photopolymerized 
 using 1-hydroxycyclohexyl phenyl ketone ( aldrich chemical 
 co. , inc . , a representation 
 of the obtained polymer network is presented in supporting information figure s1 . pegdma 
 were dissolved in deionized water at the desired concentration ( 10 , 
 20 35 , 50% w / w ) and mixed in a sonicator bath to help dissolution . the photoinitiator was then incorporated to reach a final concentration 
 of 0.075% w / w . the prepolymeric solution was transferred to 
 an amber vial and placed inside a sealed glovebox ( cole - parmer , vernon 
 hills , il ) . at this point , the glovebox chamber is sealed ( with one 
 small exit to allow air to exit ) and purged with nitrogen ( 99% pure ) . 
 after the camera was purged for approximately 30 min , a syringe needle 
 ( 20 gauge and several inches long depending on the size of the bottle ) 
 was located at the end of the tubing where nitrogen was released . 
 the needle was then placed inside the monomer solution bottle ( using 
 a septum cap for the bottle makes things easier ) making sure that 
 the nitrogen pressure was reduced enough to create a very slow bubbling 
 ( with an additional needle to provide for air to escape and avoid 
 bottle overpressure ) , which should be maintained for 20 min to remove 
 oxygen , a free radical scavenger . slow bubbling is very important 
 as it prevents the solution from forming bubbles that will eventually 
 be contained in the solid polymer . if proper removal of oxygen is 
 not achieved , polymerization will not proceed accordingly . the monomer 
 solution was then introduced within the proper container and exposed 
 to a uv light source to initiate the polymerization ( uv lamp 100 w 
 mercury arc , efo acticure , canada ) , at an intensity of 35 mw / cm . equilibrium swelling studies 
 were conducted to determine the mesh size distribution as a function 
 of the final peg gel composition . for this purpose , hydrogels were 
 prepared as described in the previous section and introduced by capillarity 
 between two microscope slides separated by a teflon spacer of 1.5 
 mm thickness . monomer mixtures ( pegma and pegdma ) were diluted to 
 obtain the following compositions ( 10 , 20 , 35 , and 50% w / w ) and polymerized 
 as described in the previous section . diameter disks , and their volume , corresponding 
 to the relaxed state ( vr ) , was determined 
 using a heptane density kit ( vogayer pro model vp114cn ohaus corporation , 
 pine brook , nj , usa ) . this technique simply uses the differences in 
 weight of an unknown sample volume and uses the known density of a 
 solvent , in this case heptane , to determine the desired volume . heptane 
 was used as solvent because it is hydrophobic , and therefore , 
 peg hydrogels do not swell ( incorporate the solvent ) . for this purpose , discs were weighed in air ( wa ) and then 
 in heptane ( wh ) to determine the relaxed 
 state volume ( vr):1where h is the known density 
 of heptane . samples were then kept in 15 ml of deionized water at 
 37 c in a water bath for 3 days . finally , the 
 membranes were dried under vacuum until no further changes in weight 
 were observed . the volume of the dried samples was then measured ( vd ) using the aforementioned heptane density 
 protocol . the measured volumes can now be correlated to the volume 
 fraction of polymer in the relaxed and swollen states ( v2,r , v2,s ) as described by23 with this information , the molecular 
 weight between cross - links , mc , 
 was calculated using eq 4 . because mn is expected 
 to be large , given the high molecular weight 
 of the monomer and the high reactivity of acrylate groups , the term 
 2/mn was neglected.4here , v is the specific 
 volume of the polymer ( 0.898 cm / s ) , v1 is the molar volume of water ( 18 cm / mol ) , 
 1 is the flory polymer water ) ,  is the number of links 
 after polymerization , 2 for vinyl polymers , and mr is the molecular weight of the monomer ( 1100 da ) . the 
 value of mc was then replaced into 
 eq 5 to determine the mesh size ( ).5the statistical analysis of the results 
 was 
 performed using minitab 15 . analysis of variance 
 ( anova ) followed by tukey s post hoc test was used for data 
 sets with multiple comparisons . a two - layer configuration system was used 
 to estimate the diffusion coefficient by mach the experimental cassette was made by separating two glass plates 
 ( 8  4 cm ) with a teflon frame of 2.4 mm thickness sandwiched 
 between the glass plates with vacuum grease to prevent solution filtration 
 from the plates . the border was sealed with bee wax , leaving a hole 
 for the injection of the solution . three different peg hydrogel matrices 
 were prepared following the aforementioned protocol , filling the 50% 
 of the volume of the cassette . after polymerization , the cassette 
 was placed in the sample arm of the mach zehnder interferometer 
 and filled with an agarose sol ( 0.5% w / v ) containing 1.7 m nacl . the 
 change in the refraction index , which is proportional to local concentration , 
 was followed every 12 s for 2 h. a phase - shift mach zehnder 
 interferometer ( ps - mzi ) based on wavelength modulation was used 
 for this purpose , following a scheme similar to the one reported by 
 ischii . in a mach zehnder experiment , a laser 
 beam is split into a reference and a sample beam . the sample beam 
 propagates through the experimental volume before recombining with 
 the reference beam . this results in the generation of interference 
 fringes ( interferogram ) at the imaging plane . the interferogram 
 carries the information on the optical path variation between the 
 varying sample beam and the still reference beam . therefore , the instrument 
 delivers a map of relative refraction index variations , calculated 
 from a set of five phase - shifted images . for this type of measurement , 
 a discrete form of fick s second law can be used in order to estimate the diffusion coefficient 
 from the interferometric phase:6where 
  is a coefficient relating the 
 refraction index to concentration and  stands 
 for the discrete laplacian operator . with this description , fick s 
 law can be verified in a well - known time and space grid and does not 
 require the a priori knowledge of starting and boundary conditions 
 nor is it necessary to define the refraction index  concentration 
 coefficient ,  , and therefore it provides local estimates . only 
 a set of two refractive index variation images taken at different 
 times ti and ti+1 are required . lysozyme from hen 
 egg white ( hewl ) ( > 90% purity ) , insulin from bovine pancreas ( > 90% 
 purity ) , sodium phosphate dibasic , tris hydrochloride , sodium 
 chloride , acetic acid glacial , and sodium acetate were purchased from 
 sigma - aldrich ( st louis , mo ) . agarose , with tf = 78 c and tg = 24 c , 
 and the granada crystallization box ( gcb ) were purchased from hampton 
 research ( 34 journey aliso viejo , ca ) . protein and crystallization 
 solution were prepared in 50 mm sodium acetate buffer ph 4.5 , filtered 
 ( 0.22 and 0.45 m , respectively ) , and stored at 4 and 20 c , 
 respectively . protein concentration was determined spectrophotometrically 
 at 280 nm using a molar extinction coefficient of 2.66 and 1.06 cm ml mg for lysozyme and insulin , 
 respectively , with a power wave biotek instruments spectrophotometer 
 ( highland park , box 998 , winooski , vt , serial no . counterdiffusion 
 experiments were set up using two configurations , the gel acupuncture 
 method ( game ) with capillaries of different diameters ( from 0.1 to 
 0.8 mm ) and the three - layer configuration ( 3l ) using capillaries of 
 2.5 mm inner diameter ( figure 1 ) . ( a ) for the game configuration , the protein 
 is mixed with the peg hydrogel and polymerized prior to the introduction 
 of the capillary in the agarose gel . ( b ) for the 3l configuration , 
 the protein  agarose or the protein  peg ( prepolymeric ) 
 solution is first loaded ( first layer ) . this was followed by the gentle addition of the prepolymeric 
 solution of peg ( 20 , 35 , or 50% w / w concentrations ) , which was also 
 polymerized by uv exposition ( second layer or plug ) . the last step 
 in both experimental setups is the addition of the crystallization 
 solution ( third layer ) . game experiments ( figure 1a ) were 
 used to 
 study the influence of peg hydrogel on the nucleation and growth of 
 lysozyme crystals . briefly , an agarose layer at 0.4% ( w / v ) concentration 
 was prepared by dissolving the right amount of agarose in the sodium 
 acetate buffer , warmed to 90 c , poured into the gcb , and allowed 
 to cool . the peg  protein mix was prepared following the described 
 protocol but using a solution of 40 mg / ml of lysozyme as the solvent 
 in the prepolymeric solution . three different pegma pegdma 
 hydrogels were prepared at 5 , 7 , and 9% ( w / w ) . the resulting mixture 
 was loaded into 0.8 mm inner diameter capillary within the glovebox 
 and exposed to the uv light . the capillaries were punctured in agarose 
 gel to a depth of approximately 10 mm . the precipitant , 1.75 m nacl 
 in 50 mm sodium acetate buffer ph 4.5 , was added and the box externally 
 sealed with parafilm . the prepolymeric mixture consisted 
 of insulin at 20 mg / ml and peg monomers . these were loaded in 0.8 
 mm inner diameter capillaries and subsequently illuminated with uv 
 light . the capillaries containing the entrapped insulin was punctured 
 in 0.2% ( w / v ) agarose layer and crystallized by adding 0.5 m sodium 
 phosphate dibasic in 0.1 m tris / hcl ph 9.0 . the 3l experiments 
 ( figure 1b ) were prepared 
 in tubes of 2.4 mm inner diameter to ( i ) study the influence of the 
 peg - based gel on the nucleation and growth of lysozyme crystals and 
 ( ii ) to study the influence of the mesh size of the plug ( second layer ) 
 on the development of the counterdiffusion pattern by following 
 the nucleation front . in the first case , experiments in 3l pegdma 
 hydrogels were prepared at a polymer concentration of 10% ( w / w ) dilution 
 using a lysozyme solution at 40 , 50 , or 60 mg / ml as solvent . the prepolymeric 
 solution was loaded into each tube ( 2.4 mm diameter ) until it reached 
 a height of 40 mm from the bottom ( first layer : protein chamber ) . then , 
 a layer of 5 mm of peg ( 50% w / w dilution ) hydrogel was poured on top 
 of it and polymerized under the uv source ( second layer : plug ) . this 
 procedure was performed with the first layer covered with aluminum 
 foil to avoid any unnecessary exposure . similar experiments 
 were conducted with agarose . in this case , 
 agarose sol was prepared at 0.4% ( w / v ) and kept at around 3540 
 c . the sol was mixed in a volume ratio of 1:1 with previously 
 prepared protein solutions at 80 , 100 , and 120 mg / ml that resulted 
 in 40 , 50 , and 60 mg the agarose / protein solution was allowed to settle until a protein 
 chamber with 0.2% ( w / v ) of agarose was obtained . then the peg ( 50% 
 w / w ) plug was polymerized as described above . finally , the crystallization 
 solution ( 1.75 m nacl in 50 mm sodium acetate buffer ph 4.5 ) was added 
 on top of the peg plug layer to fill up the additional 40 mm , which 
 constitutes the precipitant chamber ( third layer ) , to both sets of 
 experiments ( pegs and agarose ) . in the second study ( influence 
 of the mesh size ) , the protein chamber 
 was filled with 70 mg / ml lysozyme concentration gelled with either 
 10% ( w / w ) peg hydrogel or 0.2% ( w / v ) agarose . then the second layer , 
 5 mm , was prepared using peg at three different concentrations , 20 , 
 35 , and 50% ( w / w ) . finally , the crystallization solution was added 
 to the samples . in all cases , the addition of the crystallization 
 solution layer 
 was taken as the starting point . experiments were followed by measuring 
 the observable advancement of the nucleation front at 24 h intervals 
 with the help of a microscope ( see figure s2 ) . each experiment was 
 repeated three times , and the results were analyzed for any statistical 
 deviation . the nucleation front rate was obtained from the slope of 
 the linear curve fitting to the front position versus the square root 
 of time . although it is not very accurate , 
 this observable nucleation front position allows us to compare the 
 evolution of each experiment . x - ray data were collected at room 
 temperature from gel grown crystals using the 3l configuration . crystal 
 were extracted from the tube and mounted in capillaries of 0.7 mm 
 inner diameter for room temperature data collection . diffraction data 
 were recorded on a bruker smart 6000 ccd detector with kappa configuration 
 ( x8 proteum ) using cu k radiation from a bruker microstar microfocus 
 ( montel optics ) rotating - anode generator operated at 45 kv and 60 
 ma . were recorded with 30 s exposure time per frame taking 
 for each degree of oscillation and a crystal - to - detector distance 
 of 40 mm . integrated intensity information was obtained for each reflection , 
 scaled with saint and corrected for absorption with sadabs from the 
 proteum software suite ( bruker axs inc . ) . b factors 
 were obtained from the wilson plot representation carried out with 
 truncate of the ccp4 suite . in counterdiffusion experiments , 
 there are a number of parameters 
 that can be modified to control the evolution of the supersaturation , 
 provoking the precipitation / crystallization of the protein within 
 the capillary . protein and crystallization agent 
 concentrations could be the first choice , but this implies that either 
 the final equilibrium concentration can not be fixed ( if crystallization 
 agent is tuned ) or that protein concentration will have to be increased 
 ( if the crystallization agent is maintained at a constant concentration ) . 
 in order to start with similar chemical conditions , we have to modify 
 some physical properties of the system as deduced form the grashof 
 number.7where l is the characteristic 
 length of the system ( cm ) ( i.e. , the capillary diameter or the pore 
 size of the gel ) , l is the solutal 
 expansivity ( cm / mg ) , c is the 
 concentration difference , g is gravity ( cm / s ) , and v is the kinematic viscosity ( cm / s ) . the simplest and effective way to reduce grn would be to modify l , which in 
 our case is the inner diameter of the capillary or the pore size of 
 the gel . if the diameter of the capillary is altered , we will also 
 be changing the protein volume and , as a consequence , the initial 
 precipitant concentration would have to be adapted . for capillaries 
 with a diameter larger than 0.2 mm , the contribution of convection 
 to mass transport increases . in these cases , the protein solution 
 may be gelled to keep the experiment under a diffusion mass transport 
 scenario . this analysis motivated us to investigate a second option , 
 that is , the control of the evolution of the supersaturation 
 within a counterdiffusion experiment by controlling the pore 
 size of the gel matrix used in the plug . this could be a simple way 
 to avoid any possible downside effect due to the protein gel 
 interaction . to evaluate all feasible 
 operational conditions , we first determined the mesh size (  ) 
 of the proposed hydrogels at different synthesis conditions . changes 
 in the mesh size were obtained by adjusting the monomer dilution ratios 
 and calculated according to eq 4 . determination of the mesh size of various peg plug compositions 
 employed to control diffusion of the crystallization agent , obtained 
 by equilibrium swelling theory . error bars represent standard error 
 of the mean of the mesh size ( n = 4 for 10% , n = 9 for 20% , n = 8 for 35% , and n = 8 for 50% peg composition ) . statistical differences 
 are considered when p < 0.05 ; * p > 0.5 for 20 and 35% . the values of the mesh sizes calculated from swelling data 
 indicated 
 a reduction of the pore size as the concentration of the monomer in 
 the prepolymeric solution increases . although it is understood that 
 other methods can be applied to change a hydrogel s mesh size 
 ( for example , cross - linker concentration ) , for this particular polymer 
 configuration ( pegma with a peg chain containing 23 units and pegdma 
 also with peg chains of 23 units ) , it was found that using a decreased 
 amount of cross - linker under similar dilution conditions did not yield 
 a mechanically stable hydrogel ( data not shown ) . therefore , for this 
 particular application , the cross - linker concentration was maintained 
 constant at 50% molar ratio . this method of controlling the mesh size 
 has been previously used by torres - lugo and peppas who demonstrated 
 that the mesh size can also be controlled by varying the dilution 
 ratio . thus , our experiments were conducted 
 maintaining a constant monomer / cross - linker ( pegma / pegdma ) ratio , 
 only varying the dilution ratio to tune the final gel pore size . experimental results indicate that mesh size values varied from 
 approximately 42 to 10  for 10 to 50% ( w / w ) composition , respectively . 
 moreover , the mesh size values for those hydrogels with 2035% 
 peg composition did not show any significant statistical differences . 
 in our case , the difference in dilution between these two compositions 
 may be too small and measurements could fall within the margin of 
 error of the experimental protocol . however , the general trend observed 
 is that when the monomer concentration in the prepolymeric solution 
 is reduced , there is an increase of mesh size . . a clear effect 
 on the estimated diffusion coefficient of nacl was observed for all 
 polymer compositions . estimates of the diffusion coefficient of nacl in cross - linked 
 peg - based hydrogels of different compositions obtained by ps - mzi compared 
 with the free water value ( diamond ) . these results indicated that the 
 diffusion coefficient changes 
 depending on the hydrogel structure . even though the mesh size estimation 
 for peg at 20 and 35% was not statistically different , it is clear 
 that the diffusion of sodium chloride is controlled by the pore size 
 of the gel matrices . therefore , we may conclude that it is possible 
 to tune the development of supersaturation by controlling the 
 diffusion of the crystallization agents without modifying the chemical 
 composition of neither the crystallization solution nor the protein 
 solutions . gel 
 pore size in the development of supersaturation in 3l counterdiffusion 
 experiments was investigated by measuring the advancement of the nucleation 
 front as a function of time . in this setup , the protein chamber ( 70 
 mg / ml ) was gelled with agarose ( 0.2% w / v ) . the precipitant diffusion 
 was controlled by varying the pore size of the second layer ( plug 
 of 5 mm ) by employing peg hydrogels of increasing concentration . protein 
 and precipitant chambers were 40 mm in length ( see figure 1 ) . the nucleation process began approximately 
 12 h after the addition of the crystallizing agent . all experiments 
 demonstrated a typical counterdiffusion pattern , with high nucleation 
 density near the plug , which decreases along the protein chamber , 
 resulting in a lower number of crystals of larger size at the end 
 of the tube . the nucleation front position was plotted against 
 the square root of time , and in all cases , it produced linear trends , 
 which indicated that the transport of the crystallization agent was 
 dominated by passive diffusion following fick s second law . therefore , the slope of the linear curve fitting 
 is a measure of the rate at which the nucleation front is moving along 
 the protein chamber . the results depicted in figure 4 illustrate the nucleation front rate along the protein chamber , 
 that is , the slope of the linear curve fit , as a function of gel concentration 
 ( pore size ) . although in all cases crystals of millimeter 
 size were obtained at the end of the protein chamber , results indicated 
 that the 50% ( w / w ) dilution peg hydrogel plugs rendered crystals slightly 
 larger when compared to experiments with 20 or 35% ( w / w ) plugs ( figure 
 s3 ) . slope of the linear curve fit to the nucleation front position 
 vs the square root of time , for each peg plug composition ( gel pore 
 size is indicated in the right axes ) . this slope is a measure of the 
 nucleation front rate in 3l counterdiffusion experiments . error bars 
 for the mesh size represent one standard deviation from the average 
 value . we have also evaluated the compatibility 
 of peg hydrogels as potential crystallization media for protein crystallization 
 experiments . for these studies , we used the 3l and the game configurations 
 ( see description in the materials and methods ) . in previous experiments , we learned that gel concentrations higher 
 than 10% impeded crystal growth , and therefore , all experiments were 
 performed using a maximum of 10% ( w / w ) polymer concentration in the 
 protein chamber . the lowest dilution ( 5% w / w ) was determined as the 
 concentration where a solid polymeric structure could be obtained . therefore , these experiments were conducted with polymers synthesized 
 with monomer concentrations between 5 and 10% ( w / w ) . lysozyme 
 crystals were successfully grown in 0.8 mm capillaries in the presence 
 of peg hydrogels at different polymer concentrations ( i.e. , degrees 
 of monomer dilutions prior to polymerization ) . a monomer solution 
 containing a 1:1 molar ratio of monomer and cross - linker was further 
 diluted with the appropriate protein solution to obtain a monomer 
 concentration ranging from 5 to 10% ( w / w ) . crystals grown at 5 and 
 7% hydrogel concentration showed the typical shape of lysozyme crystals 
 composed of prismatic ( 110 ) and pyramidal ( 001 ) faces , while at 9% 
 ( w / w ) , crystals showed a rounded shape that became almost spherical 
 at 10% ( w / w ) ( figure 5 ) . lysozyme crystals grown 
 at constant temperature in peg hydrogels 
 with different polymer compositions : 5% ( w / w ) ( top left ) , 7% ( w / w ) 
 ( top right ) , 9% ( w / w ) ( bottom left ) , and 10% ( w / w ) ( bottom right ) . similar observations have already 
 been reported for lysozyme and 
 thaumatin crystals grown in silica gels . this morphological transition can been explained as the coupling 
 of the  berg effect ( i.e. , 
 the concentration at the corners and edges of a growing crystal is 
 higher than that at the center of the faces ; therefore , the supersaturation 
 is higher at the corners than at the center of the face , which means 
 that the edges grow more than the other regions and the polyhedron 
 shape is lost ) , as a consequence of the depletion zone generated during 
 crystal growth in gel media , and the loss of the energetic anisotropy 
 of growing faces at higher gel concentrations . protein crystals grown in gelled media are able to incorporate 
 gel fibers during their growth . the incorporation of gel fibers into the crystal have been pointed 
 as the principal source for the loss of face anisotropy due to the 
 homogenization of the surface energy . the incorporation of peg hydrogel fibers was analyzed by a dissolution 
 experiment of a lysozyme crystal grown at 9% ( w / w ) . when the supersaturation 
 is lowered , the lysozyme molecules abandon the crystal surface , making 
 visible the gel skeleton ( figure s4 ) . as in the case of agarose and 
 silica gels , peg hydrogel - grown crystals are composite materials containing 
 both the hydrogel and the protein molecules . even though protein 
 crystals incorporate the peg hydrogel , it does 
 not seem to affect crystal quality . to confirm this , we have compared 
 lysozyme crystals grown in 10% ( w / w ) peg hydrogel with crystals grown 
 in 0.2% ( w / w ) agarose . data sets were collected under identical conditions 
 ( i.e. , number of frames , exposition time , etc . ) , and the quality was 
 evaluated from standard parameters . besides 
 the typical small differences that can be found between different 
 specimens , all crystals diffracted similarly to the resolution of 
 1.85  limited by the used data collection configuration producing 
 similar statistical values ( table 1 and figure 
 s5 ) . agarose gel is known to induce nucleation , whereas silica gel is considered to inhibit the nucleation of lysozyme . to evaluate the possible effect of peg hydrogels on the nucleation 
 of lysozyme , we followed the progress of the nucleation front using 
 the 3l configuration of the counterdiffusion technique . the 
 observable nucleation front was determined as the position of the 
 last crystal observed with a microscope ( see figure s2 ) . the results 
 were also compared with identical experiments using agarose to gel 
 the protein solution . for this particular experiment , it was decided 
 to keep a constant gel concentration of agarose , 0.2% ( w / w ) , and peg , 
 10% ( w / v ) , in the protein chamber and a plug composition of 50% ( w / w ) 
 peg . figure 6a demonstrates the evolution of 
 the observable nucleation front as a function of the square root of 
 time for each protein concentration . this representation allows us 
 to conclude that a diffusional mass transport process controls the 
 nucleation front evolution within the selected time frame since it 
 follows the analytical solution of fick s law . therefore , the slope of the linear curve fitting is a measure 
 of the rate at which the nucleation front is moving along the protein 
 chamber ( figure 6b ) . experiments conducted 
 in agarose evolved as expected ; the nucleation front moved faster 
 as the protein concentration was increased . also , the three experimental 
 series ( 40 , 50 , and 60 mg / ml ) performed with agarose started at almost 
 the same time with a small delay of the experiments performed with 
 40 mg / ml lysozyme . these results indicated that the plug pore size 
 ( 50% ( w / w ) peg ) controls the diffusion of the crystallization solution , 
 and , therefore , determines the starting time . a closer 
 look at the evolution of the experiments ( figure 6a ) indicated that the experiments performed using the peg 
 hydrogel started to nucleate earlier than the experiments conducted 
 in agarose . taking into account that agarose acts as a nucleation 
 inducer , the peg hydrogel is acting as 
 a crystallization agent , enhancing the local supersaturation 
 reached by the diffusion of sodium chloride . although this effect 
 is observed for all peg hydrogel experiments , at 60 mg / ml of lysozyme 
 a delay can be observed . we believed that at high protein concentration , 
 over 50 mg / ml , the interaction between the protein molecules and the 
 peg monomers might affect the polymerization , and protein could be 
 entrapped within the gel network or even produce some protein denaturation . any of these effects could lower the supersaturation when compared 
 with the other two experimental series . second , in all experiments 
 performed with peg - based hydrogel , the nucleation front advanced at 
 a constant rate ( slope ) independently of the protein concentration 
 ( figure 6b and table s1 ) . this observed behavior 
 points to an effect of the peg pore diameter , much smaller than agarose 
 pore size . we hypothesize that the peg hydrogel is playing an essential 
 role on the control of supersaturation by limiting the diffusion 
 of protein molecules . ( a ) observable nucleation front position as a function 
 of the square 
 root of time of 3l counterdiffusion experiments . the crystallization 
 chamber contains protein , in 10% ( w / w ) peg cross - linked hydrogel or 
 0.2% ( w / v ) agarose , at three concentrations . error bars for the observable 
 nucleation front position vs the square root of time represent one 
 standard deviation from the average of three independent experiments . 
 lines ( dotted for agarose experiments ) represent the linear curve 
 fit . the slope of the fit , which gives an idea of the nucleation front 
 rate , is plotted in b vs protein concentration for the six types of 
 experiments . error bars in the slope vs protein concentration represent 
 the 95% confidence interval . finally , as a proof of concept , we have also tested the use 
 of 
 a peg hydrogel , a highly biocompatible material , as crystallization 
 media for the production of crystalline insulin peg composite 
 material . in order to minimize protein consumption , we selected the 
 gel acupuncture method setup of the counterdiffusion technique 
 to grow insulin  peg crystals in capillaries of 0.8 mm inner 
 diameter . crystals were observed after 8 days and grown along the 
 capillary to a final size of approximately 460  420  420 m ( figure 7 ) . insulin crystals grown 
 in 10% ( w / w ) peg hydrogel using the game 
 configuration with capillaries of 0.8 mm inner diameter punctuated 
 in agarose 0.2% ( w / v ) . a and b representing the lower , close to the 
 crystallization agent insertion point , and the upper parts of the 
 capillary , respectively . we have demonstrated that peg - based hydrogels 
 are good candidates 
 to produce protein crystals under reduced convection . there are two 
 possible ways to reduce the convection using peg hydrogels in counterdiffusion 
 experiments : ( i ) mixed with the protein solution or ( ii ) as a membrane 
 to control the diffusion of the crystallization agent into the protein 
 crystallization chamber . in the first case , the physicochemical 
 interactions between the macromolecule and the polymer could 
 play an important role on the control of nucleation and crystal shape . 
 as in the case of agarose and silica gels , crystals grown in peg hydrogels 
 incorporate the gel matrix , producing composite materials of both 
 the hydrogel and the protein molecules . this composite material may 
 find potential use as a controlled drug delivery system . in the second 
 case , the use of a material prepared ad hoc may allow fine - tuning 
 of the development of the supersaturation in the crystallization 
 chamber by controlling the polymer mesh size and could help to produce 
 crystals of larger size suitable for neutron diffraction .
poly(ethylene glycol ) ( peg ) hydrogels 
 are highly biocompatible 
 materials extensively used for biomedical and pharmaceutical 
 applications , controlled drug release , and tissue engineering . in 
 this work , 
 peg cross - linked hydrogels , synthesized under various conditions , 
 were used to grow lysozyme crystals by the counterdiffusion 
 technique . 
 crystallization experiments were conducted using a three - layer 
 arrangement . 
 results demonstrated that peg fibers were incorporated 
 within lysozyme crystals controlling the final crystal shape . 
 peg 
 hydrogels also induced the nucleation of lysozyme crystals to a higher 
 extent than agarose . 
 peg hydrogels can also be used at higher concentrations 
 ( 2050% w / w ) as a separation chamber ( plug ) in counterdiffusion 
 experiments . in this case , peg hydrogels control the diffusion of 
 the crystallization agent and therefore may be used to tailor the 
 supersaturation to fine - tune crystal size . as an example , insulin 
 crystals were grown in 10% ( w / w ) peg hydrogel . 
 the resulting crystals 
 were of an approximate size of 500 m .