Nucleation of Sub-Micrometer Protein Crystals in Square-Shaped

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Nucleation of Sub-micrometer Protein Crystal in Square Shape Macro-Porous Silicon Structures U. Salazar-Kuri, J. O. Estevez, E. E. Antunez, B. S. Martinez, J. B. Warren, Babak Andi, M. L. Cerniglia, V. Stojanoff, and V. Agarwal Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.5b00243 • Publication Date (Web): 04 May 2015 Downloaded from http://pubs.acs.org on May 10, 2015

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Crystal Growth & Design

Nucleation of Sub-micrometer Protein Crystal in Square Shape MacroPorous Silicon Structures U. Salazar-Kuri†‡, J. O. Estevez‡, E.E. Antunez‡, B. S. Martinez-Aguila†, J. B. Warren§, Babak Andi†, M. L. Cernigliaǁ, V. Stojanoff*†, V. Agarwal**‡. †

Photon Science Directorate, Brookhaven National Laboratory, Upton, New York 11973, USA.

‡

Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, CP 62210, Mexico 3. § ǁ

Instrumentation Division, Brookhaven National Laboratory, Upton, New York 11973, USA.

Department of Bioengineering, Binghamton University, Binghamton, NY, 13902 USA.

Corresponding Authors *Email: [email protected] **Email: [email protected] 1 ACS Paragon Plus Environment


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ABSTRACT: Macro-porous silicon substrates, with square shaped pores, have been used to crystallize hen egg white lysozyme by the sitting drop vapor diffusion method. The X-ray diffraction technique (XRD) was used to determine the tetragonal structure of the crystals. Use of asymmetric anodization procedure to produce pore size gradients in porous structure, ranging from 400 nm to 1µm, resulted in the formation of sub-micron sized protein crystals within the macroporous structure. The presence of the crystals was observed by field emission scanning electron microscopy and confirmed by Raman and Infrared spectroscopy. The present work provides an experimental evidence of sub-micron crystal growth from pore corners and rough sides of the pore walls, attributed to the reduction of the potential energy for nucleation, in accordance with the different mathematical models developed so far.

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1. INTRODUCTION Proteins are the major machinery of life, but their functionality can be understood only after the determination of the corresponding three dimensional structure. Protein crystals have shown significant benefits in the delivery of biopharmaceuticals due to the fact that crystals are the most concentrated form for drugs, and its low viscosity formulation results in the controlled delivery of proteins.1 Most of the macromolecular structure data in the protein data bank (PDB) was obtained by X-ray crystallography (80%) and the rest by nuclear magnetic resonance (NMR) (~16%), theoretical modeling (2%) and other methods.2 While X-ray diffraction and NMR provide complementary information, the most effective technique for protein structure determination is X-ray diffraction crystallography. This technique requires the fabrication of high quality protein crystals, which is often a major experimental challenge. Formation of high quality crystals implies control over their conception stage in the crystallization process (nucleation stage).3 Nucleation is the mechanism leading to the formation of clusters of molecules displaying translational and rotational order that have their own rules, different from those of crystal growth. It is well known that nucleation controls the structure of the crystallizing phase, the number of particles, and the crystal size, that appear in a crystallization system.4 On the other hand, the size of the crystals used to perform X-ray diffraction is of particular importance, due to the fact that the integrated intensity of an X-ray diffraction peak from a crystal is proportional to the ratio of its diffraction volume to its unit cell volume.5 Typically, the crystal size used for X-ray diffraction experiments is of the order of 50-500 µm. In general, as the crystal grows, it increases the tendency to accumulate defects such as stacking faults. Therefore, defects often limit the maximum size of good-quality crystals. Additionally, it is believed that many interesting protein species may only form good crystals with sub-µm dimensions.6-8 Although crystals grow in supersaturated solutions by accretion, the beginning of the nucleation process is at the zone of moderate supersaturation. Above this region protein precipitates and below, in the metastable zone, crystals may grow and no further nucleation will occur as the probability is very low. Beyond the solubility curve, i.e., undersaturation zone or the zone of equilibrium concentration, the solution is stable. 3,4,9 There are two types of nucleations: homogeneous and heterogeneous. Homogeneous nucleation is a probabilistic phenomenon which takes place in the bulk of the solution when certain numbers of molecules cluster together by the Brownian motion forming and breaking crystallites continuously until eventually they overcome a free-energy barrier to form a critical nucleus that is large enough to continue growing.4 Heterogeneous nucleation is initiated by solid particles or surfaces with nucleation-inducing properties, even if the supersaturation is insufficient for homogeneous nucleation.10 Therefore, heterogeneous nucleation offers the kinetic advantages to obtain bigger and better-order crystals.9 If the conditions are appropriate, after nucleation, freely moving molecules in the solution flow toward the formed cluster and attach to its surface.



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Although the process is conceptually easy to understand, the nucleation of a crystal from its mother solution is difficult to achieve experimentally. Nucleants are agents that induce heterogeneous nucleation of protein crystals in a controlled manner by providing a substrate on which a crystal nucleus can form and grow. Finding a universal nucleant substrate is what Chayen and Saridakis have called the "holy grail" of protein crystallography.11 Different techniques have been implemented to overcome the issue of the extremely small rate of crystal nucleation and to induce the nucleation process.3 Several materials have been used as nucleants such as minerals,12-14 horse and human hair,15,16 hydrogel membranes,17 charged surfaces such as silicon,18 polymeric films and charged glass,19-21 using protein based materials, 22-24 or porous materials.9,25 Among the porous materials used to constrain protein molecules in the pores and thereby induce them to aggregate in crystalline order, the first proposed as nucleant was porous silicon (PSi).26 The basic idea of using porous materials is to confine the protein in small volumes. Minimizing the volume of single test, maximizes the number of different conditions that can be studied with a given quantity of protein.27 PSi has attracted the attention of scientific community and inspired researchers towards optical sensing and biological applications due to its excellent properties such as high surface area (>200 m2/cm3)28, controllable pore size29, reactive surface chemistry30, its ability to allow the infiltration of chemical and biological substances, tunable optical properties31, and its biodegradable nature and biocompatibility.32 In general, PSi is a nanostructured material composed of silicon, air, and sometimes silicon dioxide, so it is considered an effective material 33 , with optical and structural features allowing the fabrication of complex photonic crystals (PCs). 34,35 Depending on the anodization parameters, i.e, electrochemical solution, type and resistivity of wafers, applied current density, several types, shapes and sizes of pores can be formed on a Si wafer. For PSi sensing applications or biofiltration, the pore size determines most of the adsorptive properties of the material.29 In protein crystal growth using porous templates, the nucleation process is related to pore size; the specific surface area of the porous surface can be increased/decreased by reducing/increasing the pore size. Therefore, control over the crystallization process can be achieved. On the other hand, PSi surfaces have been used as a nucleant material for several protein crystallizations under metastable conditions. 3, 9-11 Taking into account that the study of the nucleation process and growth of protein crystals is one of the most important and underdeveloped areas of structural biology, and encouraged by the unique features of PSi, such as controllable pore sizes, large surface area and convenient surface chemistry, macro-pore size structures offer an ideal substrate to study the heterogeneous nucleation of hen egg white lysozyme crystals.


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2. EXPERIMENTAL SECTION 2.1 Porous Silicon preparation. Macro-porous silicon substrates, used in this work, were obtained by the electrochemical etching of n-type, phosphorous doped, single-side polished, (100) oriented silicon. To study the crystal nucleation in PSi, low doped substrates (n-) with a resistivity of 8-12 Ω*cm were used. The electrolyte consisted of a mixture of aqueous 48 wt.% HF (hydrofluoric acid) and absolute ethanol (99.9%) in a volumetric ratio of 1:4, respectively. Etching was performed using a small cell made of Teflon and utilizing an experimental setup schematically represented in Figure 1a. This experimental configuration uses an electrodeassisted lateral electric and perpendicular magnetic field. Ga-In eutectic was rubbed only at the two extreme ends for each substrate (anode/cathode).36 The etching process was executed in the dark with no illumination and at room temperature for 10 min. The electric field is generated when a lateral potential Vx= 50 V is biased across the two ends of the substrate (15 mm x 30 mm), giving rise to the flow of current Ix. A magnetic field (By =20 mT) is placed perpendicular to the electric field direction, so that majority charge carriers (electrons, e-) flowing in the xr r r direction will be swept down by the effect of the resulting Lorentz force Fz = q (v x × B y ) , as indicated in the Figure 1a schematic. Generation of valence band holes at the effective area of the substrate exposed to the HF based electrolyte (i.e. HF-silicon interface) promotes the reaction. An increased magnetic field density leads to a major accumulation of valence band holes at the HF-silicon interface while on the other hand, a large lateral potential supplied will contribute to the formation of a structural gradient in the supply of holes along the electric field direction. The increase in the porosity on the PSi sample is from locations A to D as shown schematically in Figure 1b.36 Large square-shaped macro-pores were observed towards the anodic region (near to location D) while relatively small macro-pores were formed towards the cathodic region (near to location A) of the sample. This process has been described in more detail by Antunez et al.37. 2.2 Crystallization and characterization. Crystals of hen egg white lysozyme (HEWL) were grown inside as well as on the surface of the macro-porous structure by the sitting drop vapor diffusion method (Figure 1d), with a mother liquor solution of 0.1M NaOAc (Sigma Aldrich Lot. 100K0272, 99 %) pH = 4.7 and 1.71 M of NaCl (Sigma Aldrich Lot. 32K1225, 99.5 %). Protein drops were made up to 25 mg/ml lysozyme (Sigma Aldrich Lot. 027K14051, 95 %) in 0.1M NaOAc pH = 4.7, added in ratio 1:1 to the mother liquor solution. In approximately 12 h crystals with diamond shape were observed by optical microscopy. Crystals prepared under such conditions were harvested for X-ray data collection at 100 K under open-flow nitrogen cryostat. Figure 2a shows the X-ray diffraction pattern from a crystal harvested from the PSi surface. Xray diffraction experiments were carried out at beamline X6A at the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory (BNL) at X-ray energy of 13.2 keV and beam size of 150 X 150 µm. With HKL2000 program package38, HEWL crystal in Figure 2a was indexed in the space group P43212 with cell parameters a = 78.88 Å and c = 36.97 Å with a mosaicity of 0.27. Figure 2b shows the X-ray diffraction pattern of a typical PSi sample with 5 ACS Paragon Plus Environment


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lysozyme crystals under the same conditions but with a beam size of 200 x 100 µm. In the present case, the signals revealing the presence of protein crystals (small spots around the center of the image with resolution of 4.17 Å) and silicon (big spots outside the blue circle) are observed. Rings have been attributed to the overall polycrystalline characteristic 39 revealed due to the randomly oriented small protein crystallites inside the pores of the composite structure (demonstrated through scanning electron microscopy in the latter part of the manuscript). In order to perform the scanning electron microscopy (SEM) imaging, an Emitech K575X platinum sputter coater was used to deposit 3-4 nm of platinum on to the sample for avoiding any charge accumulation over the highly resistive macro PSi layer. Samples were imaged with a JEOL JSM-6500F and field emission Hitachi 4800 SEM. The acceleration voltages were 5 and 10 kV at room temperature and at a base pressure of 10-4 Pa. Samples were analyzed with single crystal Raman spectrophotometer (Horiba Jobin-Yvon Inc.) using beam line X26C at the NSLS.40 The system consists of a laser source, a Raman probe head containing an edge filter specific for one of the laser excitation energies, an iHR 550 spectrometer, and a Synapse CCD detector. Geometry of the data collection is in backscatter mode. All the connections are through optical fibers. LabSpec (Horiba-JY Inc.) and CBASS (BNL) softwares were used for data collection. 41,42 For the reported experiments, the laser excitation source was 300 mW 532 nm green laser (diode-pumped solid state (DPSS) laser (Laser Quantum)). Laser power level at the sample was approximately 35 mW and the slit size was 1000 µm. Grating was 600 gr/mm and spectral midpoint was 1158 cm-1 with the collection range of 400 – 1800 cm-1. Resolution was at 4-5 cm-1 and accumulation number and exposure time were adjusted for the optimum spectra as described for each sample. Fourier Transform Infrared (FTIR) spectra were measured with a Perkin Elmer Spotlight 400 FTIR Imaging System microscope at room temperature and solid-state white light illumination in reflection mode. The aperture was 50x50 microns. The spectrometer uses a Duet detector, a design containing a single element MCT detector and a linear array MCT imaging detector. The spectrometer was controlled from a computer using Spectrum IMAGE software.43 For each run, a total of 64 scans were collected at a resolution of 4 cm-1.

3. RESULTS AND DISCUSSION 3.1 SEM Imaging. Figure 3a shows a low magnification micrograph of the PSi surface covered by the dried droplets of the protein solution revealing the formation of protein crystals. Crystals on the surface show the typical feature of tetragonal protein crystals with well defined faces and edges (see Figure 3b).


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Figure 3c shows the boundary between the dried content from the droplet (where the large crystals are found to form) and the square-shaped macro-pores filled with tiny crystals (Figure 3d). Crystals fill the pores with dimensions of ≤100 nm and are clearly found on the walls of the larger pores exhibiting different shapes and sizes. The presence of crystals within the pores is attributed to the fractal nature of the PSi surface and pore walls.44 In order to observe the growth of the crystals along the pore depth, the sample was mechanically cleaved to observe cross-sectional view. Figures 4a and b show different regions of the cross section with different pore sizes. Figure 4a shows the growth of the protein crystals within the pores of approximately 1 µm, while 4b shows the growth inside pores with an average size of 400 nm. Figure 4b also reveals the presence of pores formed along the [001] (horizontal pores on the image) direction and not only the formation of macro-pores along [100] (vertical pores on the image) direction of the silicon substrate. These side-branched pores are of the order of 100 to 200 nm width. However, the crystals are in just a few pores and they do not fill the cavities. These samples were naturally oxidized, so the wettability of the different pores could be different and hence crystals just grow in some pores and not in the deepest pores. On the other hand, the crystals that grow in those few pores are also found in the branched pores (pores propagating along the [001] direction as shown in Figures 4c and d).

For the sake of clarity and to experimentally support this hypothesis, some PSi substrates were thermally oxidized in air at 400 ºC for ten minutes. The angle of wettability changed from around 72(1)º to 12(1)º from the naturally oxidized sample to the oxidized sample, respectively, showing the chemical modification of the surface without morphological changes (See Figure S1, Supporting information,). 45 The principal reason for the changed wetting behavior is governed by the hydrophilic nature of thin silicon oxide film formed on the surface of the complete porous structure, after oxidation. Figure 5 shows the FESEM (Field Emission SEM) cross-sectional micrographs of the oxidized samples. From Figure 5a it can be observed that pores are full of crystals grown with different sizes. Even on the regions of low crystallization density, it is possible to observe well define crystals (see Figure 5b) growing from the corners and sides in the pores. It is clear that the oxidation of the pores improved the wettability of the walls and hence increased the number of nucleation centers for protein crystallization (Figure 5c). Figure 5d reveal the crystals filling the branched pores as well. Regardless of the fact that the mechanical cleaving process could be violent enough to expel crystals from the pore walls, the differences between Figure 4 and Figure 5 are clear enough to reveal the effect of PSi oxidation i.e., an increased wettability of the pore walls and hence the creation of more points for crystal nucleation. Stolyarova and Nemirovsky,46 using the Shewmon model,47 showed how pores of conic shape reduce the volume of the critical nucleus and therefore lower the potential barrier for nucleation, provoking a faster nucleation in the pore than on the flat surface. Figures 3, 4 and 5 reveal the preferred crystal formation from the corners and



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rough sides of the pores (vertical or horizontal)48,49 and grow without any other constraint over the empty space in the pores, verifying the mathematical models proposed so far.10, 46, 48 3.2 Raman Spectroscopy. In order to eliminate the possibility of having salt crystals instead of protein crystals, Raman measurements were performed. Raman spectrum serves as a sensitive and selective fingerprint of three dimensional structure, intermolecular interactions, dynamics, and vibrations.50 Figure 6 shows the spectrum obtained from the hybrid structure formed on the naturally oxidized PSi sample and compared with the reference samples. For the sake of clarity, high intensity silicon peak at 521 cm-1 is not fully shown. Table 1 enlists all observed HEWL single crystal Raman lines. Table 1. Characteristic Raman spectrum lines of hen egg white lysozyme single crystal.51-54 frecuency (cm-1) 429 460 509 529 545 577 624 634 646 661 700 731 761 800 836 858 879 900 936 984 1006 1014 1035 1055

Assignment

ν(S-S) ν(S-S) Trp Trp Phe S-H Tyr ν(C-S) (disulfide) ν (C-S) (Methionine) Trp Tyr Tyr Trp ν (C-C) ν (C-C) Amide I' and Tyr Phe Trp Phe S=O

frecuency (cm-1) 1078 1103 1127 1162 1179 1198 1210 1240 1253 1262 1274 1280 1304 1338 1363 1432 1448 1459 1494 1553 1582 1622 1660 1670-1675 1680-1695

Assignment v(C-N) v(C-N) Trp Tyr Tyr Tyr and Phe Tyr and Phe Amide III Amide III Amide III Amide III Amide III Amide Trp Trp N-H bending vibration of the indole ring C-H, deformation vibration C-H, deformation vibration His Trp, O2 from air (partial) Trp Trp Amide I B-Sheet Disorder structures


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Observation of characteristic Raman lines from the symmetric and antisymmetric vibrations for HEWL single crystal indicates that the crystals are proteins and not salts. Most of these characteristic lines are also observed when crystals are over the PSi surface (red curve). One characteristic silicon line at 521 cm-1 and a broad band from 917 to 1000 cm-1 is observed along with a peak in the PSi-solution spectrum (blue curve) at 480 cm-1 associated with the amorphous phase Si-O interactions. Furthermore, when the laser impinges on the region far from the big crystals over the PSi surface, but inside the droplet (PSi-solution curve), it is possible to observe some of the characteristic lines of HEWL single crystals, indicating the presence of tiny HEWL crystals on the pore surface (in agreement with the SEM observations). 3.3 FTIR Spectroscopy. On the other hand, water is a strong IR-absorbing medium, and protein crystals are always in a water medium. In protein science, the effect of the environment on vibrational frequencies is often a characteristic parameter providing the information on the functioning of proteins.55 IR spectroscopy offers the complement to Raman spectroscopy on vibrational transitions of molecules. Figure 7 presents the normalized FTIR absorbance spectra of hybrid structure formed with naturally oxidized PSi-protein crystal and compared with the reference samples. In the curve corresponding to PSi-LysXtal (and Lys-Xtal), the peak at 1112 cm-1 is due to C-N stretching vibrations and C-H in-plane bending vibration assigned to Histidine (His), and the peak at 1263 cm-1 is associated to COH in-plane bending vibration assigned to Aspartic acid (Asp) amino acids in the region of the amide III. The absorption line at 1415 corresponds to NH, C-H in-plane bending vibration and C-C stretching vibration, assigned to Tryptophan (Trp) amino acid, and the peak at 1470 cm-1 is related to CH2 in-plane bending vibration, associated to Phenylalanine (Phe) amino acid in the region of the amide II. The peaks at 1565 and 1697 cm-1 are associated to Glutamic acid (Glu) COO- and Arginine (Arg) CN3H5+ amino acids, respectively in the region of the amide I.55, 56 Broad absorption band on the right reveals the presence of the amine N-H stretch at 3500-3700 cm-1,57 the carboxylic acid O-H stretch (25003000 cm-1) and the also C-H vibrations at 2850-3000 cm-1.58 Similar to Raman spectroscopy, the signal from the lysozyme can be observed even in the region far from the clear presence of a crystal, indicating the presence of tiny crystals on the pore surface.

4. SUMMARY AND CONCLUSION In conclusion, square macro-porous silicon structures with gradient in pore size, along the direction of the applied EF, have been used as heterogeneous nucleation material for protein crystals due to the confinement of molecules within the pore corners and rough surfaces of the pore walls. A wide range of pore sizes, from 100 to 1000 nm can be obtained on the same structure and all serve as nucleation centers to grow sub-micron different sized protein crystals within the pores. Oxidized substrates show an increased nucleation rate due to an increase in the 9 ACS Paragon Plus Environment


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hydrophilic nature of the PSi surface. Mathematical models predicted this behavior in micropores; additionally, if we consider the self-similar property of PSi, the same models can be applied for meso and macro-pores. The functionalizing of porous materials opens the possibility of their use as templates for the crystallization of sub-micron sized proteins and, when developed as photonic crystals, to be used to monitor real-time nucleation process. ASSOCIATED CONTENT Supporting Information Differences in the angle of wettability. This information is available free of charge via internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Email: [email protected] **Email: [email protected] Notes The authors declare no competing financial interest

ACKNOWLEGMENTS U. S. K. thanks the postdoctoral fellowship from CONACyT (No. 208147) and also special thanks to the National Institute of General Medical Science (NIGMS) supporting during the research stay at BNL. X6A beam line was supported by the NIGMS of the National Institute of Health (NIH) under agreement GM-0080. The NSLS, Brookhaven National Laboratory is supported by the US Department of Energy (DOE) under contract No. DE-AC02-98CH10886. JEOL JSM-6500F SEM, Department of Instrumentation and at the Center for Functional Nanomaterials, Brookhaven National Laboratory. FE-SEM Hitachi 4800, Center for Functional Nanomaterials, Brookhaven National Laboratory, which is supported by the U.S. Department of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886


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BA was supported by the NIGMS (P41GM103473) of the NIH and the US DOE of Biological and Environmental Research (FWP BO-70). Spectroscopic data were collected at beamline X26C of the National Synchrotron Light Source (NSLS). Use of the NSLS, Brookhaven National Laboratory, was supported by the U.S. DOE, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886. Special thanks to Dr. Ruth Pietri and Ramonita Diaz who assisted SM on IR spectroscopy measurements.

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Figure Captions: Figure 1. Schematics for the fabrication of the macro-porous 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 macro-pores 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. 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 macro-pores showing different features of the tetragonal structure. SEM images of as formed macroporous silicon have been reported by Antunez et al.36,37 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 crystals growth also on the branched pores formed as structural feature of this kind of porous structures.

Figure 5. Cross section FESEM micrographs of wafers oxidized at 400 ºC. (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. Figure 6. Raman spectra for naturally oxidized PSi. Image taken from the PSi region where the droplet of the protein solution to prepare crystals is (PSi-solution) placed, one HEWL crystal over the PSi surface (PSi-LysXtal) and of one single HEW Lysozyme crystal (Lys-Xtal). Inset shows the lysozyme crystal spectrum in the region of the characteristic fingerprint for lysozyme. Figure 7. FTIR spectra of the hybrid structure (PSi- LysXtal) formed on the naturally oxidized PSi. Reference samples (bare PSi, protein crystal and PSi-protein solution) are shown for comparison.



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Figure 1


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Figure 2



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Figure 3


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Figure 5


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Figure 7


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For Table of Contents use only Nucleation of Submicrometer Protein Crystal in Square Shape Macro-Porous Silicon Structures U. Salazar-Kuri , J. O. Estevez , E.E. Antunez , B. S. Martinez-Aguila , J. B. Warren , Babak Andi , M. L. Cerniglia , V. Stojanoff* , V. Agarwal** . †‡

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Photon Science Directorate, Brookhaven National Laboratory, Upton, New York 11973, USA.

†

Centro de Investigación en Ingeniería y Ciencias Aplicadas, UAEM, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos, CP 62210, Mexico .

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Instrumentation Division, Brookhaven National Laboratory, Upton, New York 11973, USA.

Department of Bioengineering, Binghamton University, Binghamton, NY, 13902 USA.

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Macro-porous silicon substrates, with square shaped pores, have been used to crystallize hen egg white lysozyme by the sitting drop vapor diffusion method. Demonstration of sub-micron crystal growth from pore corners and rough sides of the pore walls is attributed to the reduction of the potential energy for nucleation, in accordance with the different mathematical models developed so far.


Nucleation of Sub-Micrometer Protein Crystals in Square-Shaped

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