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Article

pubs.acs.org/crystal

1 Nucleation of Sub-Micrometer Protein Crystals in Square-Shaped

2 Macroporous Silicon Structures

3 U. Salazar-Kuri,†,‡ J. O. Estevez,‡ E. E. Antunez,‡ B. S. Martinez-Aguila,† J. B. Warren,§ Babak Andi,†

∥ ,†
4 M. L. Cerniglia, V. Stojanoff,* and V. Agarwal*,‡
†
5 Photon Science Directorate and §Instrumentation Division, Brookhaven National Laboratory, Upton, New York 11973, United
6 States
‡
7 Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos CP
8 62210, Mexico
∥
9 Department of Bioengineering, Binghamton University, Binghamton, New York 13902, United States
10 *
S Supporting Information

11 ABSTRACT: Macroporous silicon substrates, with square-shaped

12 pores, have been used to crystallize hen egg white lysozyme by the
13 sitting drop vapor diffusion method. The X-ray diffraction
14 technique was used to determine the tetragonal structure of the
15 crystals. Use of an asymmetric anodization procedure to produce
16 pore size gradients in porous structure, ranging from 400 nm to 1
17 μm, resulted in the formation of sub-micrometer-sized protein
18 crystals within the macroporous structure. The presence of the
19 crystals was observed by field emission scanning electron
20 microscopy and confirmed by Raman and infrared spectroscopy. The present work provides experimental evidence of sub-
21 micrometer crystal growth from pore corners and rough sides of the pore walls, attributed to the reduction of the potential
22 energy for nucleation, in accordance with the different mathematical models developed so far.

1. INTRODUCTION from a crystal is proportional to the ratio of its diffraction 49

23 Proteins are the major machinery of life, but their functionality volume to its unit cell volume.5 Typically, the crystal size used 50

24 can be understood only after the determination of the for X-ray diffraction experiments is on the order of 50−500 μm. 51

25 corresponding three-dimensional structure. Protein crystals In general, as the crystal grows, it increases the tendency to 52

26 have shown significant benefits in the delivery of biopharma- accumulate defects such as stacking faults. Therefore, defects 53

27 ceuticals because crystals are the most concentrated form for often limit the maximum size of good-quality crystals. 54

28 drugs, and its low viscosity formulation results in the controlled Additionally, it is believed that many interesting protein species 55

29 delivery of proteins.1 Most of the macromolecular structure may only form good crystals with sub-micrometer dimen- 56

30 data in the protein data bank (PDB) was obtained by X-ray sions.6−8 57

31 crystallography (80%) and the rest by nuclear magnetic Although crystals grow in supersaturated solutions by 58

32 resonance (NMR) (∼16%), theoretical modeling (2%), and accretion, the beginning of the nucleation process is at the 59

33 other methods.2 While X-ray diffraction (XRD) and NMR zone of moderate supersaturation. Above this region protein 60

34 provide complementary information, the most effective precipitates and below, in the metastable zone, crystals may 61

35 technique for protein structure determination is XRD grow, and no further nucleation will occur as the probability is 62

36 crystallography. This technique requires the fabrication of very low. Beyond the solubility curve, i.e., undersaturation zone 63

37 high quality protein crystals, which is often a major or the zone of equilibrium concentration, the solution is 64

38 experimental challenge. Formation of high quality crystals stable.3,4,9 65

39 implies control over their conception stage in the crystallization There are two types of nucleations: homogeneous and 66

40 process (nucleation stage).3 Nucleation is the mechanism heterogeneous. Homogeneous nucleation is a probabilistic 67

41 leading to the formation of clusters of molecules displaying phenomenon that takes place in the bulk of the solution when 68
42 translational and rotational order that have their own rules, certain numbers of molecules cluster together by the Brownian 69
43 different from those of crystal growth. It is well known that motion forming and breaking crystallites continuously until 70
44 nucleation controls the structure of the crystallizing phase, the eventually they overcome a free-energy barrier to form a critical 71
45 number of particles, and the crystal size that appear in a
46 crystallization system.4 On the other hand, the size of the Received: February 17, 2015
47 crystals used to perform XRD is of particular importance Revised: April 8, 2015
48 because the integrated intensity of an X-ray diffraction peak

© XXXX American Chemical Society A DOI: 10.1021/acs.cgd.5b00243

Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

Figure 1. Schematics for the fabrication of the macroporous silicon structure and protein crystals growth within its pores. (a) Electrode-assisted
experimental setup, (b) photograph of the PSi sample, (c) schematic of the sample with a pore size gradient in the size of the square-shaped
macropores and its side-branching, (d) setup for the protein crystallization by sitting drop technique, and (e) protein crystals growth inside the pores
of the structure.

72 nucleus that is large enough to continue growing.4 Heteroge- nanostructured material composed of silicon, air, and some- 110
73 neous nucleation is initiated by solid particles or surfaces with times silicon dioxide, so it is considered an effective material,33 111
74 nucleation-inducing properties, even if the supersaturation is with optical and structural features allowing the fabrication of 112
75 insufficient for homogeneous nucleation.10 Therefore, hetero- complex photonic crystals (PCs).34,35 Depending on the 113
76 geneous nucleation offers the kinetic advantages to obtain anodization parameters, i.e., electrochemical solution, type 114
77 bigger and better-order crystals.9 If the conditions are and resistivity of wafers, applied current density, several types, 115
78 appropriate, after nucleation, freely moving molecules in the shapes and sizes of pores can be formed on a Si wafer. For PSi 116
79 solution flow toward the formed cluster and attach to its sensing applications or biofiltration, the pore size determines 117
80 surface. Although the process is conceptually easy to under- most of the adsorptive properties of the material.29 In protein 118
81 stand, the nucleation of a crystal from its mother solution is crystal growth using porous templates, the nucleation process is 119
82 difficult to achieve experimentally. related to pore size; the specific surface area of the porous 120
83 Nucleants are agents that induce heterogeneous nucleation surface can be increased/decreased by reducing/increasing the 121
84 of protein crystals in a controlled manner by providing a pore size. Therefore, control over the crystallization process can 122
85 substrate on which a crystal nucleus can form and grow. be achieved. On the other hand, PSi surfaces have been used as 123
86 Finding a universal nucleant substrate is what Chayen and a nucleant material for several protein crystallizations under 124
87 Saridakis have called the “holy grail” of protein crystallog- metastable conditions.3,9−11 125
88 raphy.11 Taking into account that the study of the nucleation process 126
89 Different techniques have been implemented to overcome and growth of protein crystals is one of the most important and 127
90 the issue of the extremely small rate of crystal nucleation and to underdeveloped areas of structural biology, and encouraged by 128
91 induce the nucleation process.3 Several materials have been the unique features of PSi, such as controllable pore sizes, large 129
92 used as nucleants such as minerals,12−14 horse and human surface area and convenient surface chemistry, macropore size 130
93 hair,15,16 hydrogel membranes,17 charged surfaces such as structures offer an ideal substrate to study the heterogeneous 131
94 silicon,18 polymeric films and charged glass,19−21 using protein- nucleation of hen egg white lysozyme (HEWL) crystals. 132
95 based materials,22−24 or porous materials.9,25 Among the
96 porous materials used to constrain protein molecules in the 2. EXPERIMENTAL SECTION
97 pores and thereby induce them to aggregate in crystalline order, 2.1. Porous Silicon Preparation. Macroporous silicon substrates, 133
98 the first proposed as nucleant was porous silicon (PSi).26 The used in this work, were obtained by the electrochemical etching of n- 134
99 basic idea of using porous materials is to confine the protein in type, phosphorus doped, single-side polished, (100) oriented silicon. 135
100 small volumes. Minimizing the volume of a single test To study the crystal nucleation in PSi, low doped substrates (n−) with 136
101 maximizes the number of different conditions that can be a resistivity of 8−12 Ω*cm were used. The electrolyte consisted of a 137
102 studied with a given quantity of protein.27 mixture of aqueous 48 wt % HF (hydrofluoric acid) and absolute 138
103 PSi has attracted the attention of the scientific community ethanol (99.9%) in a volumetric ratio of 1:4, respectively. Etching was 139

104 and inspired researchers toward optical sensing and biological performed using a small cell made of Teflon and utilizing an 140
experimental setup schematically represented in Figure 1a. This 141 f1
105 applications due to its excellent properties such as high surface experimental configuration uses an electrode-assisted lateral electric 142
106 area (>200 m2/cm3),28 controllable pore size,29 reactive surface and perpendicular magnetic field. Ga−In eutectic was rubbed only at 143
107 chemistry,30 its ability to allow the infiltration of chemical and the two extreme ends for each substrate (anode/cathode).36 The 144
108 biological substances, tunable optical properties,31 and its etching process was executed in the dark with no illumination and at 145
109 biodegradable nature and biocompatibility.32 In general, PSi is a room temperature for 10 min. The electric field is generated when a 146

B DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

Figure 2. X-ray diffraction pattern of (a) one crystal harvested from PSi surface and (b) PSi wafer with lysozyme crystals. At the center there are
small spots revealing the presence of the protein crystals and also large black spots coming from the silicon wafer.

Figure 3. SEM micrographs of (a) HEWL crystals grew over the surface of the porous silicon sample, (b) lysozyme protein crystals showing
characteristic tetragonal features with well-defined faces and edges. (c) The interface between the accumulated protein on the surface (formation of
the large crystals) and the remaining porous structure with infiltrated protein. (d) Magnified view of the pores with sizes ranging from approximately
1 μm to less than 100 nm. Protein crystals were found at different depths inside all macropores showing different features of the tetragonal structure.
SEM images of the as-formed macroporous silicon have been reported by Antunez et al.36,37

147 lateral potential Vx= 50 V is biased across the two ends of the substrate on the other hand, a large lateral potential supplied will contribute to 157
148 (15 mm × 30 mm), giving rise to the flow of current Ix. A magnetic the formation of a structural gradient in the supply of holes along the 158
149 field (By = 20 mT) is placed perpendicular to the electric field electric field direction. The increase in the porosity on the PSi sample 159
150 direction, so that majority charge carriers (electrons, e−) flowing in the is from locations A to D as shown schematically in Figure 1b.36 Large 160
151 x-direction will be swept down by the effect of the resulting Lorentz square-shaped macropores were observed toward the anodic region 161
(near location D), while relatively small macropores were formed 162
152 force Fz⃗ = q(vx⃗ × By⃗ ), as indicated in Figure 1a schematic. Generation
toward the cathodic region (near location A) of the sample. This 163
153 of valence band holes at the effective area of the substrate exposed to process has been described in more detail by Antunez et al.37 164
154 the HF-based electrolyte (i.e., HF−silicon interface) promotes the 2.2. Crystallization and Characterization. Crystals of HEWL 165
155 reaction. An increased magnetic field density leads to a major were grown inside as well as on the surface of the macroporous 166
156 accumulation of valence band holes at the HF-silicon interface, while structure by the sitting drop vapor diffusion method (Figure 1d), with 167

C DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

Figure 4. Cross section FESEM micrographs after protein crystallization in an as-etched macro PSi. (a) and (b) Regions with crystals inside the
pores at different scales. Image reveals the presence of crystals in some pores. However, (c) and (d) show crystal growth also on the branched pores
formed as a structural feature of this kind of porous structure.

168 a mother liquor solution of 0.1 M NaOAc (Sigma-Aldrich Lot. NSLS.40 The system consists of a laser source, a Raman probe head 202
169 100K0272, 99 %) pH = 4.7 and 1.71 M of NaCl (Sigma-Aldrich Lot. containing an edge filter specific for one of the laser excitation 203
170 32K1225, 99.5%). Protein drops were made up to 25 mg/mL energies, an iHR 550 spectrometer, and a Synapse CCD detector. 204
171 lysozyme (Sigma-Aldrich Lot. 027K14051, 95 %) in 0.1 M NaOAc pH Geometry of the data collection is in backscatter mode. All the 205
172 = 4.7, added in a 1:1 ratio to the mother liquor solution. In connections are through optical fibers. LabSpec (Horiba-JY Inc.) and 206
173 approximately 12 h crystals with diamond shape were observed by CBASS (BNL) software was used for data collection.41,42 For the 207
174 optical microscopy. Crystals prepared under such conditions were reported experiments, the laser excitation source was 300 mW 532 nm 208
175 harvested for X-ray data collection at 100 K under open-flow nitrogen green laser (diode-pumped solid state (DPSS) laser (Laser 209
f2 176 cryostat. Figure 2a shows the XRD pattern from a crystal harvested Quantum)). Laser power level at the sample was approximately 35 210
177 from the PSi surface. XRD experiments were carried out at beamline mW, and the slit size was 1000 μm. Grating was 600 gr/mm and 211
178 X6A at the National Synchrotron Light Source (NSLS) at Brookhaven spectral midpoint was 1158 cm−1 with the collection range of 400− 212
179 National Laboratory (BNL) at X-ray energy of 13.2 keV and beam size 1800 cm−1. Resolution was at 4−5 cm−1, and the accumulation 213
180 of 150 × 150 μm. With HKL2000 program package,38 HEWL crystal number and exposure time were adjusted for the optimum spectra as 214
181 in Figure 2a was indexed in the space group P43212 with cell described for each sample. 215
182 parameters a = 78.88 Å and c = 36.97 Å with a mosaicity of 0.27. Fourier Transform Infrared (FTIR) spectra were measured with a 216
183 Figure 2b shows the X-ray diffraction pattern of a typical PSi sample PerkinElmer Spotlight 400 FTIR Imaging System microscope at room 217
184 with lysozyme crystals under the same conditions but with a beam size temperature and solid-state white light illumination in reflection mode. 218
185 of 200 × 100 μm. In the present case, the signals revealing the The aperture was 50 × 50 μm. The spectrometer uses a Duet detector, 219
186 presence of protein crystals (small spots around the center of the a design containing a single element MCT detector, and a linear array 220
187 image with resolution of 4.17 Å) and silicon (big spots outside the blue MCT imaging detector. The spectrometer was controlled from a 221
188 circle) are observed. Rings have been attributed to the overall computer using Spectrum IMAGE software.43 For each run, a total of 222

189 polycrystalline characteristic39 revealed due to the randomly oriented 64 scans were collected at a resolution of 4 cm−1. 223

190 small protein crystallites inside the pores of the composite structure
191 (demonstrated through scanning electron microscopy in the latter part 3. RESULTS AND DISCUSSION
192 of the manuscript). 3.1. SEM Imaging. Figure 3a shows a low magnification 224 f3
193 In order to perform the scanning electron microscopy (SEM) micrograph of the PSi surface covered by the dried droplets of 225
194 imaging, an Emitech K575X platinum sputter coater was used to
the protein solution revealing the formation of protein crystals. 226
195 deposit 3−4 nm of platinum onto the sample to avoid any charge
196 accumulation over the highly resistive macro PSi layer. Samples were
Crystals on the surface show the typical feature of tetragonal 227

197 imaged with a JEOL JSM-6500F and field emission Hitachi 4800 SEM. protein crystals with well-defined faces and edges (see Figure 228

198 The acceleration voltages were 5 and 10 kV at room temperature and a 3b). 229
199 base pressure of 10−4 Pa. Figure 3c shows the boundary between the dried content 230
200 Samples were analyzed with single crystal Raman spectropho- from the droplet (where the large crystals are found to form) 231
201 tometer (Horiba Jobin-Yvon Inc.) using beamline X26C at the and the square-shaped macropores filled with tiny crystals 232

D DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

Figure 5. Cross-section FESEM micrographs of wafers oxidized at 400 °C. (a) Pores filled of crystals, revealing the increment of centers of
nucleation when PSi walls are oxidized. (b) Amplification of one region with low density of crystal formation but still with well-formed crystals. (c)
Pores with many crystals with different sizes and crystalline habits. (d) Branched pores full of protein crystals.

233 (Figure 3d). Crystals fill the pores with dimensions of ≤100 nm hydrophilic nature of thin silicon oxide film formed on the 264
234 and are clearly found on the walls of the larger pores exhibiting surface of the complete porous structure, after oxidation. Figure 265 f5
235 different shapes and sizes. The presence of crystals within the 5 shows the field emission SEM (FESEM) cross-sectional 266 f5
236 pores is attributed to the fractal nature of the PSi surface and micrographs of the oxidized samples. From Figure 5a it can be 267
237 pore walls.44 observed that pores are full of crystals grown with different 268
238 In order to observe the growth of the crystals along the pore sizes. Even on the regions of low crystallization density, it is 269
239 depth, the sample was mechanically cleaved to observe the possible to observe well-defined crystals (see Figure 5b) 270
f4 240 cross-sectional view. Figure 4a,b shows different regions of the growing from the corners and sides in the pores. It is clear that 271
241 cross section with different pore sizes. Figure 4a shows the the oxidation of the pores improved the wettability of the walls 272
242 growth of the protein crystals within the pores of approximately and hence increased the number of nucleation centers for 273
243 1 μm, while Figure 4b shows the growth inside pores with an protein crystallization (Figure 5c). Figure 5d reveals the crystals 274
244 average size of 400 nm. Figure 4b also reveals the presence of filling the branched pores as well. 275
245 pores formed along the [001] (horizontal pores on the image) Regardless of the fact that the mechanical cleaving process 276
246 direction and not only the formation of macropores along could be violent enough to expel crystals from the pore walls, 277
247 [100] (vertical pores on the image) direction of the silicon
the differences between Figures 4 and 5 are clear enough to 278
248 substrate. These side-branched pores are on the order of 100 to
reveal the effect of PSi oxidation, i.e., an increased wettability of 279
249 200 nm width. However, the crystals are in just a few pores, and
they do not fill the cavities. These samples were naturally the pore walls and hence the creation of more points for crystal 280
250
251 oxidized, so the wettability of the different pores could be nucleation. Stolyarova and Nemirovsky,46 using the Shewmon 281

252 different, and hence crystals just grow in some pores and not in model,47 showed how pores of conic shape reduce the volume 282

253 the deepest pores. On the other hand, the crystals that grow in of the critical nucleus and therefore lower the potential barrier 283

254 those few pores are also found in the branched pores (pores for nucleation, provoking a faster nucleation in the pore than 284
255 propagating along the [001] direction as shown in Figure 4c,d). on the flat surface. Figures 3−5 reveal the preferred crystal 285
256 For the sake of clarity and to experimentally support this formation from the corners and rough sides of the pores 286
257 hypothesis, some PSi substrates were thermally oxidized in air (vertical or horizontal)48,49 and grow without any other 287
258 at 400 °C for 10 min. The angle of wettability changed from constraint over the empty space in the pores, verifying the 288
259 around 72(1)° to 12(1)° from the naturally oxidized sample to mathematical models proposed so far.10,46,48 289
260 the oxidized sample, respectively, showing the chemical 3.2. Raman Spectroscopy. In order to eliminate the 290
261 modification of the surface without morphological changes possibility of having salt crystals instead of protein crystals, 291
262 (see Figure S1, Supporting Information).45 The principal Raman measurements were performed. Raman spectrum serves 292
263 reason for the changed wetting behavior is governed by the as a sensitive and selective fingerprint of three-dimensional 293

E DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

294 structure, intermolecular interactions, dynamics, and vibra- Table 1. Characteristic Raman Spectrum Lines of Hen Egg
295 tions.50 White Lysozyme Single Crystal51−54
f6 296 Figure 6 shows the spectrum obtained from the hybrid
frequency frequency
297 structure formed on the naturally oxidized PSi sample and (cm−1) assignment (cm−1) assignment
429 1078 v(C−N)
460 1103 v(C−N)
509 ν(S−S) 1127 Trp
529 ν(S−S) 1162 Tyr
545 Trp 1179 Tyr
577 Trp 1198 Tyr and Phe
624 Phe 1210 Tyr and Phe
634 S−H 1240 amide III
646 Tyr 1253 amide III
661 ν(C−S) 1262 amide III
(disulfide)
700 ν(C−S) 1274 amide III
(methionine)
731 1280 amide III
761 Trp 1304 amide
800 1338 Trp
836 Tyr 1363 Trp
858 Tyr 1432 N−H bending vibration of
the indole ring
879 Trp 1448 C−H, deformation vibration
Figure 6. Raman spectra for naturally oxidized PSi. Image taken from 900 ν(C−C) 1459 C−H, deformation vibration
the PSi region where the droplet of the protein solution to prepare 936 ν(C−C) 1494 His
crystals is (PSi-solution) placed, one HEWL crystal over the PSi 984 amide I′ and 1553 Trp, O2 from air (partial)
surface (PSi-LysXtal) and of one single HEW lysozyme crystal (Lys- Tyr
Xtal). Inset shows the lysozyme crystal spectrum in the region of the 1006 Phe 1582 Trp
characteristic fingerprint for lysozyme. 1014 Trp 1622 Trp
1035 Phe 1660 amide I
298 compared with the reference samples. For the sake of clarity, a 1055 SO 1670−1675 B-sheet
299 high intensity silicon peak at 521 cm−1 is not fully shown. 1680−1695 disorder structures
t1 300 Table 1 shows all observed HEWL single crystal Raman lines.
301 Observation of characteristic Raman lines from the
302 symmetric and antisymmetric vibrations for HEWL single
303 crystal indicates that the crystals are proteins and not salts.
304 Most of these characteristic lines are also observed when
305 crystals are over the PSi surface (red curve). One characteristic
306 silicon line at 521 cm−1, and a broad band from 917 to 1000
307 cm−1 is observed along with a peak in the PSi-solution
308 spectrum (blue curve) at 480 cm−1 associated with the
309 amorphous phase Si−O interactions. Furthermore, when the
310 laser impinges on the region far from the big crystals over the
311 PSi surface, but inside the droplet (PSi-solution curve), it is
312 possible to observe some of the characteristic lines of HEWL
313 single crystals, indicating the presence of tiny HEWL crystals
314 on the pore surface (in agreement with the SEM observations).
315 3.3. FTIR Spectroscopy. On the other hand, water is a
316 strong IR-absorbing medium, and protein crystals are always in
317 a water medium. In protein science, the effect of the
318 environment on vibrational frequencies is often a characteristic
319 parameter providing the information on the functioning of Figure 7. FTIR spectra of the hybrid structure (PSi-LysXtal) formed
320 proteins.55 IR spectroscopy offers the complement to Raman on the naturally oxidized PSi. Reference samples (bare PSi, protein
f7 321 spectroscopy on vibrational transitions of molecules. Figure 7 crystal, and PSi-protein solution) are shown for comparison.
322 presents the normalized FTIR absorbance spectra of hybrid
323 structure formed with naturally oxidized PSi-protein crystal and corresponds to N−H, C−H in-plane bending vibration and C− 331
324 compared with the reference samples. C stretching vibration, assigned to tryptophan (Trp) amino 332
325 In the curve corresponding to PSi-LysXtal (and Lys-Xtal), acid, and the peak at 1470 cm−1 is related to CH2 in-plane 333
326 the peak at 1112 cm−1 is due to C−N stretching vibrations and bending vibration, associated with phenylalanine (Phe) amino 334
327 C−H in-plane bending vibration assigned to histidine (His), acid in the region of the amide II. The peaks at 1565 and 1697 335
328 and the peak at 1263 cm−1 is associated with COH in-plane cm−1 are associated with glutamic acid (Glu) COO− and 336
329 bending vibration assigned to aspartic acid (Asp) amino acids in arginine (Arg) CN3H5+ amino acids, respectively, in the region 337
330 the region of the amide III. The absorption line at 1415 of the amide I.55,56 Broad absorption band on the right reveals 338

F DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

339 the presence of the amine N−H stretch at 3500−3700 cm−1,57 98CH10886. Special thanks to Dr. Ruth Pietri and Ramonita 395
the carboxylic acid O−H stretch (2500−3000 cm−1), and the Diaz who assisted S.M. on IR spectroscopy measurements.

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340 396
341 C−H vibrations at 2850−3000 cm−1.58 Similar to Raman
342 spectroscopy, the signal from the lysozyme can be observed REFERENCES 397
343 even in the region far from the clear presence of a crystal, (1) Sekhon, B. S. T. J. Pharm. Sci. 2010, 34, 1−19. 398
344 indicating the presence of tiny crystals on the pore surface. (2) http://www.rcsb.org/pdb/static.do?p=general_ 399
information%5Cabout_pdb%5Cnature_of_3d_structural_data.html. 400
4. SUMMARY AND CONCLUSION (3) Chayen, N. E. Curr. Opin. Struct. Biol. 2004, 5, 577−583. 401
(4) Ruiz, J. M. J. Struct. Biol. 2003, 142, 22−31. 402
345 In conclusion, square macroporous silicon structures with a (5) Hunter, M. S.; Deponte, D. P.; Shapiro, D. A.; Kirian, R. A.; 403
346 gradient in pore size, along the direction of the applied EF, have Wang, X.; Starodub, D.; Marchesini, S.; Weierstall, U.; Doak, R. B.; 404
347 been used as heterogeneous nucleation material for protein Spence, J. C.; Fromme, P. Biophys. J. 2011, 100, 198−206. 405
348 crystals due to the confinement of molecules within the pore (6) Hunter, M. S.; Fromme, P. Methods 2011, 55, 387−404. 406
349 corners and rough surfaces of the pore walls. A wide range of (7) Bogan, M. J. Anal. Chem. 2013, 85, 3464−3471. 407
350 pore sizes, from 100 to 1000 nm, can be obtained on the same (8) Nannenga, B. L.; Gonen, T. Curr. Opin. Struct. Biol. 2014, 27, 408
351 structure, and all serve as nucleation centers to grow sub- 24−31. 409
(9) McPherson, A. Crystallization of Biological Macromolecules; Cold 410
352 micrometer different sized protein crystals within the pores.
Spring Harbor Laboratory Press: Woodbury, NY, 1999. 411
353 Oxidized substrates show an increased nucleation rate due to an (10) Chayen, N. E.; Saridakis, E.; Sear, R. P. Proc. Natl. Acad. Sci. 412
354 increase in the hydrophilic nature of the PSi surface. U.S.A. 2006, 103 (3), 597−601. 413
355 Mathematical models predicted this behavior in micropores; (11) Saridakis, E.; Chayen, N. E. Trends Biotechnol. 2008, 27 (2), 99− 414
356 additionally, if we consider the self-similar property of PSi, the 106. 415
357 same models can be applied for meso- and macropores. The (12) McPherson, A.; Shlichta, P. Science 1988, 239, 385−387. 416
358 functionalizing of porous materials opens the possibility of their (13) Kimble, W. L.; Paxton, T. E.; Rousseau, R. W.; Sambanis, A. J. 417
359 use as templates for the crystallization of sub-micrometer-sized Cryst. Growth. 1998, 187, 268−276. 418
360 proteins and, when developed as photonic crystals, to be used (14) Falini, G.; Fermani, S.; Conforti, G.; Ripamonti, A. Acta 419
361 to monitor real-time nucleation process. Crystallogr., Sect. D: Biol. Crystallogr. 2002, 58, 1649−1652. 420

■
(15) D’Arcy, A.; Mac Sweeney, A.; Haber, A. Acta Crystallogr., Sect. 421
D: Biol. Crystallogr. 2003, 59, 1343−1346. 422
362 ASSOCIATED CONTENT (16) Georgieva, D. G.; Kuil, M. E.; Oosterkamp, T. H.; Zandbergen, 423
363 *
S Supporting Information H. W.; Abrahams, J. P. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2007, 424
364 Differences in the angle of wettability. The Supporting 63, 564−570. 425
(17) Profio, G. D.; Polino, M.; Nicoletta, F. P.; Belviso, B. D.; 426
365 Information is available free of charge on the ACS Publications
Caliandro, R.; Fontananova, E.; Filpo, G. D.; Cursio, E.; Drioli, E. Adv. 427
366 website at DOI: 10.1021/acs.cgd.5b00243.

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Funct. Mater. 2014, 24, 1582−1590. 428
(18) Sanjoh, A.; Tsukihara, T.; Gorti, S. J. Cryst. Growth. 2001, 234, 429
367 AUTHOR INFORMATION 618−628. 430
368 Corresponding Authors (19) Fermani, S.; Fallini, G.; Minnucci, M.; Ripamonti, A. J. Cryst. 431
Growth. 2001, 224, 327−334. 432
369 *(V.S.) E-mail: stojanof@bnl.gov. (20) Tsekova, D.; Popova, S.; Nanev, C. Acta Crystallogr., Sect. D: 433
370 *(V.A.) E-mail: vagarwal@uaem.mx. Biol. Crystallogr. 2002, 58, 1588−1592. 434
371 Notes (21) Nanev, C. N. Cryst. Growth Des. 2007, 7, 1533−1540. 435
372 The authors declare no competing financial interest. (22) Pechkova, E.; Nicolini, C. J. Cell. Biochem. 2002, 85, 243−251. 436

■
(23) Pechkova, E.; Nicolini, C. J. Cell. Biochem. 2004, 91, 1010−1020. 437
(24) Pechkova, E.; Roth, S. V.; Burghammer, M.; Fontani, D.; Riekel, 438
373 ACKNOWLEDGMENTS C.; Nicolini, C. J. Synchrotron Radiat. 2005, 12, 713−716. 439
374 U.S.K. thanks the postdoctoral fellowship from CONACyT (25) Khurshid, S.; Saridakis, E.; Govada, L.; Chayen, N. E. Nat. 440
375 (No. 208147) and also special thanks to the National Institute Protoc. 2014, 9 (7), 1621−1633. 441
376 of General Medical Science (NIGMS) supporting during the (26) Chayen, N. E.; Saridakis, E.; El-Bahar, R.; Nemirovsky, Y. J. Mol. 442
377 research stay at BNL. X6A beamline was supported by the Biol. 2001, 312, 591−595. 443

378 NIGMS of the National Institute of Health (NIH) under (27) Arrondo, J. L. R.; Alonso, A. Advanced Techniques in Biophysics; 444
Springer Series in Biophysics; Springer: Berlin, Germany, 2006; 10. 445
379 agreement GM-0080. The NSLS, Brookhaven National (28) Herino, R.; Bornchil, G.; Barla, K.; Bertrand, C.; Ginoux, J. L. J. 446
380 Laboratory is supported by the U.S. Department of Energy Electrochem. Soc. 1987, 134, 1994−2000. 447
381 (DOE) under Contract No. DE-AC02-98CH10886. JEOL (29) Canham, L. T. Properties of Porous Silicon; DERA, Malvern, UK; 448
382 JSM-6500F SEM, Department of Instrumentation and at the INSPEC, The Institution of Electrical Engineers: London, United 449
383 Center for Functional Nanomaterials, Brookhaven National Kingdom, 1997. 450
384 Laboratory. FE-SEM Hitachi 4800, Center for Functional (30) Cullis, A. G.; Canham, L. T.; Calcott, P. D. J. Appl. Phys. 1997, 451
385 Nanomaterials, Brookhaven National Laboratory, which is 82, 909−965. 452
386 supported by the U.S. Department of Energy, Office of Basic (31) Ghulinyan, M.; Oton, C. J.; Bonetti, G.; Gaburro, Z.; Pavesi, L. J. 453
387 Energy Sciences, under Contract No. DE-AC02-98CH10886. Appl. Phys. 2003, 93, 9724−9729. 454
388 B.A. was supported by the NIGMS (P41GM103473) of the (32) Ghoshal, S.; Mitra, D.; Roy, S.; Majumder, D. D. Sens. 455
Transducers 2010, 113, 1−17. 456
389 NIH and the U.S. DOE of Biological and Environmental (33) Pap, A. E.; Kordas, K.; Vahakangas, J.; Uusimaki, A.; Leppavouri, 457
390 Research (FWP BO-70). Spectroscopic data were collected at S.; Pilon, L.; Szatmari, S. Opt. Mater. 2006, 506−513. 458
391 beamline X26-C of the National Synchrotron Light Source (34) Estevez, J. O.; Arriaga, J.; Méndez Blas, A.; Agarwal, V. Appl. 459
392 (NSLS). Use of the NSLS, Brookhaven National Laboratory, Phys. Lett. 2008, 93, 191915. 460
393 was supported by the U.S. DOE, Office of Science, Office of (35) Lin, V. S. Y.; Motesharei, K.; Dancil, K. P. S.; Sailor, M. J.; 461
394 Basic Energy Sciences, under Contract No. DE-AC02- Ghadiri, M. R. Science 1997, 278, 840−843. 462

G DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX
 Crystal Growth & Design Article

463 (36) Antunez, E. E.; Estevez, J. O.; Campos, J.; Basurto-Pensado, M.

464 A.; Agarwal, V. Physica B 2014, 453, 34−39.
465 (37) Antunez, E. E.; Campos, J.; Basurto-Pensado, M. A.; Agarwal, V.
466 Nanoscale Res. Lett. 2014, 9, 512.
467 (38) Otwinowski, Z.; Minor, W. Processing of X-ray Diffraction Data
468 Collected in Oscillation Mode, Methods in Enzymology, 276;
469 Macromolecular Crystallography, Part A, Carter, C.W., Jr., Sweet, R.
470 M., Eds.; Academic Press: New York, 1997; pp 307−326.
471 (39) Von Dreele, R. B. J. Appl. Crystallogr. 2007, 40, 133−143.
472 (40) Stoner-Ma, D.; Skinner, J. M.; Schneider, D. K.; Cowan, M.;
473 Sweet, R. M.; Orville, A. M. Synchrotron Radiat. 2011, 18, 37−40.
474 (41) Skinner, J. M.; Sweet, R. M. Acta Crystallogr., Sect. D: Biol.
475 Crystallogr. 1998, 54, 718−725.
476 (42) Skinner, J. M.; Cowan, M.; Buono, R.; Nolan, W.; Bosshard, H.;
477 Robinson, H. H.; Héroux, A.; Soares, A. S.; Schneider, D. K.; Sweet, R.
478 M. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2006, 62, 1340−1347.
479 (43) Spectrum Image Software; PerkinElmer, Inc.: Waltham, MA,
480 1998−2014.
481 (44) Bessueille, F.; Dugas, V.; Vikulov, V.; Cloarec, J. P.; Souteyrand,
482 E.; Martin, J. R. Bios. Bioel. 2005, 21, 908−916.
483 (45) Stolyarova, S.; Baskin, E.; Nemirovsky, Y. J. Cryst. Growth. 2012,
484 360, 131−133.
485 (46) Stolyarova, S.; Nemirovsky, Y. ECS Trans. 2011, 33 (16), 137−
486 145.
487 (47) Shewmon, P. G.Transformation in Metals; McGraw-Hill: New
488 York, 1969.
489 (48) Page, A. J.; Sear, R. P. Phys. Rev. Lett. 2006, 97, 065701.
490 (49) Liu, Y. X.; Wang, X. J.; Lu, J.; Ching, C. B. J. Phys. Chem. B
491 2007, 111, 13971−13978.
492 (50) Thomas, G., Jr. J. Annu. Rev. Biophys. Biomol. Struct. 1999, 28,
493 1−27.
494 (51) Lord, R. C.; Yu, N. T. J. Mol. Biol. 1970, 50, 509−524.
495 (52) Kocherbitov, V.; Latynis, J.; Misiunas, A.; Barauskas, J.; Niaura,
496 G. J. Phys. Chem. B 2013, 117, 4981−4992.
497 (53) Harada, I.; Takamatsu, T.; Tasumi, M.; Lord, R. C. Biochem.
498 1982, 21 (15), 3674−3677.
499 (54) Lambert, J. B.; Shurvell, H. F.; Cooks, R. G. Introduction to
500 Organic Spectroscopy; Macmillan: New York, 1987.
501 (55) Barth, A. Biochem. Biophys. Acta 2007, 1767, 1073−1101.
502 (56) Vasita, R.; Katti, D. S. Int. J. Nanomed. 2012, 7, 61−71.
503 (57) Zou, B.; Liu, Y.; Luo, x.; Chen, F.; Guo, X.; Li, X. Acta Biomater.
504 B 2012, 8, 1576−1585.
505 (58) Liltorp, K.; Marechal, Y. Biopolymers 2005, 79, 185−196.

H DOI: 10.1021/acs.cgd.5b00243
Cryst. Growth Des. XXXX, XXX, XXX−XXX