Preparation and Characterisation of 3D Nanotemplates for Protein Crystallisation

Umang V. Shah1, Jose V. Parambil1,2, Daryl R. Williams1, Steve J. Hinder3, Jerry Y. Y. Heng1*

1Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, United Kingdom

2Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117576

3 The Surface Analysis Laboratory, Faculty of Engineering & Physical Sciences, University of Surrey, Guildford, Surrey GU2 7XH, United Kingdom.

Tel. Number: +44 (0) 207 594 0784 E-mail address: jerry.heng@imperial.ac.uk

*To whom correspondence should be addressed.

Abstract

Heterogeneous template nucleants are gaining pace as a favoured tool for crystallisation of

proteins that maybe otherwise difficult to crystallise. Systematic understanding on protein-

nucleant interactions has to be developed to enable development of nucleants for wide

spectrum of biological macromolecules. Thorough characterisation of the nucleants is the key

starting point to achieve this aim. This report focuses on the method to produce and

characterise functionalised 3D nanotemplates with controlled porosity in the range of 3-22nm

and surface chemistry that can vary from highly hydrophilic to highly hydrophobic in nature.

BET and TEM are used to study porosity and pore size distribution while contact angle, XPS

and zeta potential are used to investigate surface chemistry of the nucleants. These

functionalised 3D nanotemplates are hereby reported to produce protein crystals

(concanavalin A and catalase) of different habits without changing any other crystallisation

parameters other than the surface chemistry of the templates. This emphasises the potential of

3D nanotemplates with well-ordered porosity and chemistry for further development in

protein crystallisation experiments.

KEYWORDS: 3D Nanotemplates, Surface Chemistry, XPS, Pore Diameter, Protein Crystal

Habits

1. Introduction

Protein crystallisation holds the promise of providing a cheaper alternative to expensive

chromatographic separation process [1]. Development of a reliable, fast and efficient protein

crystallisation methodology is widely anticipated in the biochemical market sector to reduce

the cost of future products [1-4]. Also, crystallisation of proteins can significantly help drug

development by solving the structural properties of drug targets, thereby reducing the

development time for new drugs [5]. However, protein crystallisation is a complex process

where crystallisation attempts are largely driven by trial and error instead of systematic

understanding of the crystallisation process [6].

Application of heterogeneous nucleants for protein crystallisation to promote and control

crystal nucleation is a rapidly developing field of research. Many different heterogeneous

nucleants have been applied for crystallisation of protein, list ranges from equine hair to

molecularly imprinted polymers [7-14]. Furthermore, many of the reported works are

focused on obtaining protein single crystals for structural determination and hence interested

in developing a nucleant which can work with range of proteins [11].

To develop protein crystallisation as an alternative to chromatographic separations, it is

essential to prepare protein specific templates, which allows separation of target protein from

protein mixture. To achieve this aim, the first step forward is to establish the physico-

chemical property relationship between protein and nucleant. Heterogeneous nucleation is

postulated to be a surface driven phenomenon [8]. Hence, for understanding the effects of

nucleant on specific protein, it is essential to elucidate the surface properties of nucleant and

protein. Detailed characterisation of nucleant surface properties is necessary to provide an

insight into protein-nucleant interactions. However, most of the literature on using

heterogeneous nucleants for protein crystallisation has highlighted the application of

nucleants more than the characterisation itself. Subsequently, it is difficult to highlight

definitive characteristic trends in protein-nucleant interactions due to uncertainties in nucleant

properties covering different reports [9-13, 15]. Nonetheless, comparing the various results in

using heterogeneous nucleants for protein crystallisation, the imperative parameters of

nucleant surface that aid crystallisation is considered to be surface chemistry and/or surface

porosity. Following this, our recent studies have demonstrated a correlation between protein

hydrodynamic diameter, surface chemistry and nucleant surface pore diameter which can

govern protein crystallisation [16, 17]. Once the correlation between the physico-chemical

properties of proteins and nucleants are established, the second step towards achieving the

aim of developing crystallisation as an alternative to chromatographic separation is the ability

to tailor surface properties of nucleants as per requirements.

This study aims to concentrate on the production and characterisation of 3D nanotemplates

with specific porosity and surface chemistry that can be utilised in protein crystallisation.

With this study, we focused on providing the surface characterisation of these heterogeneous

nucleants used for protein crystallisation. We hereby report the methodology to prepare 3D

nanotemplate surfaces with specific pore diameter (3-22nm range), narrow pore size

distribution and controlled surface chemistry (from highly hydrophilic to hydrophobic in

nature). Characterisation of these templates is conducted used BET, TEM, contact angle, XPS

and zeta potential. Furthermore, an application of these templates for controlling protein

crystal habit is demonstrated using catalase and concanavalin A proteins.

2. Experimental Methods

2.1 Synthesis of Particles

2.1.1 Synthesis of 3D nanotemplates with pore diameter 3.5±1.0nm

In order to tune porosity in the low pore diameter range (typically 3-4nm), a sol-gel method

was used [18]. Acidic catalyst was used to promote the hydrolysis reaction. The reactant to

catalyst molar ratios used for the synthesis was 12 – 155: 1: 0.026 – 6 (H2O: TEOS: 2N

HNO3). In a typical synthesis, HNO3 was added to deionised water (DI) and stirred for 30

minutes. TEOS was added in the acid water mixture and the solution was stirred until the

solution become clear. The sol was transferred to a glass mould, sealed and aged at 60oC for

72 hours in a vacuum oven. Aged samples were dried at 130oC for 48 hours in a vacuum

oven. Before drying, the seal of the mould is removed. Solid transparent glass discs were

obtained as a result of drying and were sintered in a tube furnace at 600oC under a N2 gas

environment.

2.1.2 Synthesis of 3D nanotemplates with pore diameter 5.5±1.5nm – 11.0±3.0nm

To synthesise the 3D nanotemplates with larger pore diameters, two different sacrificial

substrates, tri-block copolymer surfactants (Pluronic F127 & Pluronic P123), were used.

Inorganic salt, potassium chloride (KCl) was used to stabilise the structure. Also, different

acid catalysts, e.g. HCl and HNO3, were used [19, 20]. In a typical synthesis, 0.5gm of

Pluronic F127 and 2.5gm of KCl were added to 30mL 2M HCl. The mixture is stirred at

room temperature until the surfactant and inorganic salt were completely dissolved in the

acidic solution. Silica precursor, TEOS (2.08gm) was added to a solution. The solution was

stirred for 24 hours at room temperature. The sol so obtained was subjected to hydrothermal

aging at 120oC under autogenous pressure for 48 hours. Hydrothermally aged samples were

collected and dried under ambient conditions. Dried samples were sintered in a tube furnace

for 5 hours at 550oC under a N2 gas environment to remove the organic templates from the

material prepared.

2.1.3 Synthesis of 3D nanotemplates with pore diameter 16.0±3.0nm – 22.0±5.0nm

Micelle swelling agents were used in addition to tri-block copolymer sacrificial substrates, in

order to increase pore diameter in excess of 11.0nm. The organic solvents, 1,3-trimethyl

benzene (TMB) and m-xylene were used as micelle swelling agents in this part of the study.

In a typical synthesis all the processing steps and reaction mixture as described in section

2.1.2 is used, along with the addition of 0.7mL of TMB or 3.5mL of m-xylene

2.2 Surface Functionalisation of 3D Nanotemplates

The protocol proposed by K. M. Kemner et al. was adopted and optimised for surface

functionalisation of 3D nanotemplates prepared [21]. In a typical experiment, 4.5 gm of the

mesoporous silica was suspended in 125 mL of water. The mixture was continuously refluxed

at 100oC for 4 hours. After cooling to room temperature, hydrated silica particles were

suspended in 250mL toluene and stirred at room temperature for 2 hours followed by addition

of 7.5mL silane solution. Post addition of the silane solution, the mixture was refluxed for 24

hours at 100oC. After cooling to room temperature, the reaction mixture was filtered and

washed with ethanol or toluene at least 5 times to remove any unreacted silanes and dried

under vacuum at 110oC for 4 hours. Materials were stored in a sealed glass bottle at ambient

conditions. Conditions for grafting different functional groups can be obtained from the

literature [16].

2.3 Characterisation of Mesoporous Surfaces

2.3.1 N2 Sorption Measurements

Approximately 200 – 300mg of 3D nanotemplate samples was conditioned under helium

purge at 120oC for 24 hours. A fully automated MicromeriticsTristar 3000 (Micromeritics,

Norcross, USA) system was used for the measurement of isotherms at -195.8oC. The surface

area and pore size distributions were calculated using the BET and BJH methods respectively

based on the isotherm obtained using the Micromeritics Analysis Software (Micromeritics,

Norcross, USA).

If applied over a wide range of relative pressures and appropriate model selected for pore size

distribution analysis, N2 adsorption can provide information on size distributions for micro,

meso and macro-porous materials. N2 adsorption with BJH model for pore size

determination, is useful for determining pore size distributions of mesoporous and

macroporous (partially) size ranges (2-50nm). The Horvath-Kawazoe (HK) model (for slit

shaped pores) and Saito-Foley (SF) model (for cylindrical shaped pores) can be used for

determining pore size distributions in microporous region.

2.3.2 Transmission Electron Microscopy (TEM)

The transmission electron micrographs obtained in this study was imaged with a JEOL 2010

STEM (JEOL Ltd., Japan) system operated at 200kV. All transmission electron micrographs

presented in this study are bright field images and processed with the JEOL software.

2.3.3 X-ray Photoelectron Spectroscopy (XPS)

XPS spectra were obtained on all 3D nanotemplate surfaces using a Thermo VG Scientific

Theta Probe X-ray Photoelectron Spectrometer (ThermoFisher Scientific, East Grinstead,

U.K.). In this study, the twin anode AlKα X-ray source with an energy of hv=1486.6eV was

used at 15kV × 20mA (300W). All survey spectra were collected at pass energy of 200eV,

whereas all high resolution core spectra were collected at a pass energy of 20eV. After

removal of the non-linear (Shirley) background, high resolution core level spectra were used

for the quantification of the functional end groups present on the surface. The peak fitting

function used is Gaussian - Lorentzian product function. The high resolution data was fitted

using the Avantage software (Thermo VG Scientific, East Grinstead, U.K) which

incorporates sensitivity factors and corrects for the electron energy analyser transmission

function.

2.3.4 Contact Angle Measurements

A Krüss Drop Shape Analyser DSA 10 (Krüss GmbH, Hamburg, Germany) was used for the

static sessile drop contact angle measurement. Analytical grade water was used as the probe

liquid and the measurements were carried out at ambient conditions. The shape of the droplet

was fitted to obtain the contact angle using the Drop Shape Analysis software (DSA version

1.0) (Krüss GmbH, Hamburg, Germany). A minimum of 5 droplets on 3 different samples of

the same surface chemistry was measured.

2.4 Protein Crystallisation

Crystallisation of proteins was carried out by the hanging drop vapour diffusion

crystallisation method. Metastable crystallisation conditions for model protein (catalase and

concanavalin A) systems under investigation were obtained from the literature [9]. Protein

crystallisation plates were observed using an Olympus BX51 M reflective microscope

(Olympus UK Ltd., UK), and protein crystal images were taken with an Olympus DP70

digital camera (Olympus UK Ltd., UK). Single crystal X-ray diffraction (Rigaku Single

Crystal X-ray Diffractometer, Rigaku Corporation, USA) was used as confirmation tool for

protein crystals obtained.

3. Results and Discussion

3.1. Preparation and characterisation of mesoporous 3D nanotemplates

As mentioned in the experimental section 2.1, synthesis of mesoporous 3D nanotemplates

was carried out at different conditions to understand the effect of catalyst concentration,

sacrificial templates and swelling agents. The results for these experiments and

characterisation of the produced mesoporous material are discussed in this session.

3.1.1. Effect of catalyst concentration

Varying the molar ratio of silica precursor to nitric acid during synthesis of mesoporous silica

resulted in changes to the textural parameters of the resulting porous material as shown in

Table-1. Analysis of the specific surface area, total pore volume and average pore diameter

of particles showed that a concentration change from 0.03M to 0.3M nitric acid resulted only

in a marginal change to the particles with respect to their surface area and pore size. But

increasing the nitric acid further to 1.2M resulted in significant increase in average pore size

(Figure-1) along with a reduction in the specific surface area. As the acid concentration was

increased from 0.3M to 1.2M, the BET N2 isotherm also shifts from type I isotherm to type II

isotherm suggesting a transition from microporous to mesoporous adsorption regime [22].

This phenomenon can be attributed to the pore formation mechanism, which includes

condensation, hydrolysis and drying. Condensation and hydrolysis are both catalysed by acid

but exhibits reduced condensation rate near isoelectric point of colloidal silica (pH≈1-2) due

to the difference in surface charge distribution. Below the isoelectric point the condensation

reaction is faster, resulting in more aqueous by product and thereby resulting in high pore

volume and pore size [23]. The pH range measured for 0.03, 0.3 and 1.2 M nitric acid

solutions were around 1.6, 0.8 and 0.2 respectively and hence resulted in a corresponding

increase in condensation rate at high nitric acid concentration. Thus, by altering the catalyst

concentration in the suitable range, we are able to control the pore diameter in the range of

3.5±1.0nm along with a narrow pore size distribution.

3.1.2. Effect of sacrificial substrates

Mesoporous materials with pore diameter greater than 5nm has been reported in the literature,

prepared using sacrificial organic template based approach [19, 20]. A systematic approach

for using mesoporous material for protein crystallisation will require surfaces with different

pore diameters in the range of 3 – 22 nm based on the hydrodynamic radius of protein

molecules used [17]. Poly (alklyene oxide) tri block copolymers with different molecular

weights and block ratios were selected after careful consideration of the range of pore

diameters that can be obtained using the same family of surfactants. PluronicP123 and

PluronicF127 surfactants resulted in increasing the pore size to around 5.5±1.5nm and

11.0±3.0nm respectively with a narrow size distribution as shown in Figure 2. Furthermore,

TEM images show that the surfaces prepared using sacrificial templates are mesoporous with

regular 3D arrays of pores in a one dimensional channel (Figure 3(a)). The pore diameters

and the well ordered arrangements can be attributed to the micelles formed during the

process. The details on the process of micelle formation and the effect of various process

parameters on micelle structure have been widely discussed in literature and hence will not be

discussed further in this report [19, 20, 24, 25]. However, it is to be stated that the pore

diameter is directly correlated to the hydrophobic core diameter of the micelles, which

corresponds to 7nm and 15nm for P123 and F127 respectively.

3.1.3. Effect of swelling agents

Swelling agents used along with surfactants affects the micelle structure by appearing to

solubilise the surfactants to a small or moderate extend. They could increase the micelle

diameter to as high as 300% of its original size (without swelling agents) [26]. Although

swelling agents help to increase the micelle size, excessive solubilisation can result in the

disruption in micelle structure and cause disordered pores. Two common swelling agents,

TMB and m-xylene were used along with PluronicF127 to increase the pore size from

11.0±3.0nm that can be obtained without the use of swelling agents. Results in Table-1 show

that the pore diameter was increased to around 16.0±3.0nm and 22.0±5.0nm by using TMB

and m-xylene respectively (Figure 3(b) & Figure 4). Thus, the use of m-xylene was able to

result in a 40% larger pore structure without losing the ordered structural arrangement.

However, using m-xylene with Pluronic P123 resulted in a disordered pore structure due to

uncontrollable solubilisation of P123 micelles. This should be caused due to the larger

hydrophobic domain (approx. 70%) in P123 which results in large uptake of m-xylene into

micelle structure and causing its disruption (Figure 5).

Combining the above preparation and characterisation of porous material, it can be

ascertained that a precise control of the pore diameter in the range of 3nm to 22nm along with

a narrow pore size distribution and ordered structure has been achieved by controlling

catalyst concentration, use of sacrificial templates and swelling agents. These 3D

nanotemplates now form the basis for further chemical modification and use as nucleation

templates for protein crystallisation.

3.2. Characterisation of functionalised mesoporous 3D nanotemplates

The mesoporous silica templates prepared through the previous processes was functionalised

through silanisation. Contact angle, XPS and zeta potential analysis of these surfaces are then

carried out to confirm functional changes of the surface and to gain better understanding of

the chemical nature. While contact angle and zeta (ξ) potential analysis can be considered as

qualitative measurement of surface properties, XPS provides quantitative measurement of

chemical nature of the surface. Hence these three measurements provide complementary

information that can help in providing a better understanding on the effect of these surfaces

on further application.

3.2.1. Untreated 3D nanotemplate

The contact angle of water on untreated silica surface shows a very low value of 11.3°±2.8°

(Table-2) pointing to the very hydrophilic nature of the surface. The XPS measurement on

this surface exhibits Si2p and O1s spectra as expected on a silica substrate. The Si2p spectra

can be fitted only with one peak centred at 103.3eV, representative of Si-O bond, both in Si-

O-Si bond in silica as well as Si-OH bond on surface. Using the AlKα source XPS used in

this study, it would be difficult to resolve Si-O bonds in the Si-O-Si network in silica and Si-

OH groups on the surface. However, O1s spectra can be deconvoluted into two resolved

peaks at 532.3eV and 534.1eV corresponding to O-Si bond in silica and physisorbed water on

surface. In addition to the Si2p and O1s spectra, a very weak C1s peak was also observed on

untreated surface. This can be attributed to the presence of any small organic contamination

during the preparation or storage of the samples. Surface charge determined through ξ

potential measurement at pH 7 is found to be highly negative, at about -35.8mV due to the

dissociation of protons from the silanol functional groups at pH ≥ 3.0. This too corroborates

with the presence of hydroxyl groups on the surface of untreated mesoporous nanotemplates.

The hydroxyl functional groups on the untreated surface is key for further functionalisation

process since the silanes will be attached to the silica surface by silanol groups reacting with

them.

3.2.2. Functionalised 3D nanotemplates

Mesoporous material silianised with amine, chloro, phenyl and dodecyl functional silanes

were characterised using the same techniques to confirm silanisation and to measure the

resultant changes in surface properties. The static contact angle on amine functionalised

surface was approximately 3x higher than untreated surface at 33.2°±4.1°. XPS survey

spectra showed peaks at binding energy corresponding to C1s (Figure 6), N1s (Figure 7),

O1s (Figure 8) and Si2p (Figure 9), along with a very low amount of fluorine which can be

attributed to the contamination in the reagents used for silanisation or the adhesive tape used

for sample preparation. Deconvolution of the high resolution C1s spectra contains for

different peaks centred at 284.3eV, 285.0eV, 285.8eV and 287.9eV binding energies which

can be assigned to C-Si, C-Hx/C-C, C-NH2 and C-CF bonds. Except for the C-CF peak, all

other three functionalities can be attributed to the silane. The peak area ratio of the different

carbon functionalities is significant since they correspond to the atomic ratio of different

elemental states of carbon present on the surface. The peak ratio as obtained from the high

resolution XPS spectra is about 1:1:1 (C-Si:C-C:C-NH2) and is in good correlation with the

chemical structure of the propyl chain of silane where there is one carbon bonded to silicon,

one central carbon in the propyl hydrocarbon chain and one carbon attached to amine

functional group. The deconvoluted Si2p spectra shows two different peaks, centred at

102.3eV and 103.5eV, associated with Si-C and Si-O functionalities respectively with a

significantly larger peak of Si-O correlated to the contribution from the bulk Si-O-Si network

in silica. The O1s spectra can be fitted with one peak centred at a binding energy of 532.6eV,

which relates to O-Si linkage on the templates. As expected for an amine surface, the high

resolution spectra of N1s provided a symmetrical Gaussian peak centred at 399.5eV. This

peak is characteristic to NH2-C (amine) functional group present in the silane. ξ-potential

measurement at pH 7.0 showed a highly positive charge of 44.9mV, suggesting the presence

of protonated groups on the surface. This observation can be explained by the fact that the

amine group can be protonated to form NH3+ species at pH ≤ 7.0 and hence confirms

presence of free amine head groups on the functionalised surface.

On the chloro functionalised surface, the static contact angle of water was found to be 7x that

of untreated surface with a value of 82.6°±3.5°. The XPS survey spectrum exhibited the

presence of C1s, Cl2p, O1s and Si2p binding energies on the surface. The deconvoluted C1s

spectra showed three peaks at 284.4eV, 285.1eV and 286.8eV, assigned to C-Si, CHx/C-C

and C-Cl, with an area ratio of 1:1:1 as mentioned before. High resolution spectra of Cl2p

revealed the presence of Cl-C group with a binding energy at 200.0eV (Figure 10) [27]. An

additional peak at 201.6eV is also observed and can be correlated to the spin orbit splitting of

Cl2p lines (Cl2p3/2 and Cl2p1/2) which is a characteristic behaviour of surfaces containing

chlorine functional groups. The intensity ratio of Cl2p3/2:Cl2p1/2 is 1.3, which is as reported in

literature[28, 29]. The characteristics of O1s and Si2p spectra on further functionalised

surfaces that are similar to those observed on amine functionalised surface will not be

elaborated further in this report. On phenyl functionalised surface, the high resolution C1s

spectrum can be deconvoluted into three peaks with binding energies centred at 284.2eV,

284.7eV and 286.5eV. The peaks at 284.2eV and 284.7eV can be assigned to the

C(aromatic)-Si and C-C(aromatic) functionalities. The peak at 286.5eV suggests the presence

of C-O functionality on the surface, which can arise from the presence of isolated silane

molecules or partially reacted silane molecules on the organo-silane layer. It is also notable

that the 1:5 area ratio between C(aromatic)-Si and C-C(aromatic) peaks are in good

correlation with the expected silane structure on surface. Also, the deconvolution of the Si2p

spectra on phenyl functionalised surface shows two different functionalities, Si-O and Si-

C(aromatic) with associated binding energies of 102.3eV and 103.6eV. The contact angle

measurement on phenyl surface is 109.1°±4.9°, almost 10x higher than the untreated surface.

On the dodecyl functionalised surface, the surface energy is expected to be the lowest among

those reported here due to the long hydrocarbon chain without any further hydrophilic

functional group. This is confirmed by the highest contact angle with a value of 128.6°±3.2°.

The high resolution C1s spectra obtained through XPS can be deconvoluted into three peaks

centred at 284.3eV, 285.0eV and 286.5eV, which are assigned to C-Si, C-C and C-O

functionalities. The atomic ratio of C-C to C-Si functionalities was found to be about 11:1,

confirming the structure of 12 carbon long chain of dodecyl with one of the end carbon

attached to silicon atom silanol group. Another interesting observation with the dodecyl

functionalised surface was the near neutral, but slightly negative ξ-potential value of -4.7 at

pH 7.0. Since it is not possible to form carbon anion in aqueous solution at pH 7.0, it is

proposed that the small negative charge is due to the preferential adsorption of negative ions

on the surface of dodecyl functionalised surface. This assertion is supported by previous

findings that cations can be more easily hydrated and retained in the solution, resulting in

weakly negative or neutral ξ-potential values [30].

3.3. Protein crystallisation using functionalised mesoporous 3D nanotemplates

Mesoporous 3D nanotemplates are efficient heterogeneous nucleating surfaces for protein

crystallisation. The ability of 3D nanotemplates with narrow pore size distribution and

different surface chemistries in crystallising proteins even from very low concentrations was

demonstrated in of our previous reports [16]. But the effect of functionalised 3D

nanotemplates on protein crystallisation is not limited to nucleating crystals of protein which

are otherwise difficult to crystallise or in nucleating protein crystals at low supersaturation.

They can guide the protein crystallisation process to produce crystals of different habits that

are not observed unless significant changes in crystallisation parameters such as pH, salt

concentration or solvent are performed. Here we investigate the ability of functionalised 3D

nanotemplates to produce different habits of concanavalin A and catalase proteins with

molecular weights of 106kDa and 232kDa respectively. Both concanavalin A and catalase are

model proteins extensively used in crystallisation studies with well reported crystallisation

conditions.

3.3.1. Concanavalin A crystallisation using functionalised 3D nanotemplates

Concanavalin A crystallisation was achieved on 3D nanotemplates with average pore

diameter of 11.03.0nm, correlated to the hydrodynamic radius of the protein [17]. To

investigate the effect of functionalised templates on crystal habits, control experiments were

conducted on untreated 3D nanotemplates with the same pore diameter and porosity.

Functionalised surfaces with different surfaces chemistries were utilised for crystallisation.

Results from crystallisation using phenyl, dodecyl and chloro functionalised 3D

nanotemplates which resulted in significant changes in the crystal habit are discussed here.

On untreated nanotemplates, concanavalin A formed elongated tetragonal crystals as shown

in Figure 11(a), which is the most commonly observed habit of concanavalin A under the

crystallisation conditions used in this study. Phenyl functionalised templates resulted in

similar habit with shorter tetragonal crystal shape Figure 11(b). On dodecyl functionalised

templates, concanavalin A resulted in round shaped rhombic tetrahedron and chloro

functionalised surface resulted in very small but well faceted crystals as observed in Figure

11(c) & Figure 11(d) respectively. These habits of concanavalin A has been reported

previously, but were obtained by varying temperature, pH, type of salt and protein

concentration [31].

3.3.2. Catalase crystallisation using functionalised 3D nanotemplates

Similar to concanavalin A, catalase was also crystallised using 3D nanotemplates with an

average pore diameter of 11.03.0nm. On untreated mesoporous templates and phenyl

functionalised templates, bipyramidal crystal morphology was observed for catalase crystals

(Figure 12(a) & Figure 12(b)). On dodecyl functionalised surfaces, a mixture of bipyramidal

and rhombohedral habits was observed as in Figure 12(c). Under the identical conditions,

crystallisation on chloro functionalised nanotemplates resulted in very thin planar crystals

(Figure 12(d)) that exhibited a slight spherulitic growth pattern.

It is to be noted that single crystal X-ray diffraction studies were conducted on these protein

crystals to ensure that they were not salt crystals and to establish the structure of the crystals

with different habits. The results from single crystal X-ray diffraction suggests that all habits

of concanavalin A crystals reported here are the orthorhombic crystal form with space group

I222 and catalase crystals are orthorhombic crystals with P212121 space group.

To the best of our knowledge, this is the first time that different habits of concanavalin A and

catalase are produced without altering the four major solution conditions for crystallisation,

viz. pH, temperature, protein concentration and type and concentration of the precipitant

solution, which are crucial for protein crystallisation [6, 31]. In order to ensure that the effect

observed here is solely due to the combined effect of porosity and surface chemistry, various

controls were performed, i.e. the crystallisation experiments were carried out using the non-

porous glass slide silanised with the same functional groups to isolate the effect of chemistry,

crystallisation trials were placed using the protein and precipitant solution of the same batch

as well as from the different batch to eliminate the effect of variation in preparation of

solution, the surfaces applied were properly degassed or surfaces without cleaning were used

for the experiments to verify the effect of the surface impurities. It was observed that the

crystals formed in these control experiments did not show any effect on habit and there was

no crystals formed when non-porous templates even with the same surface chemistry was

used, thereby corroborating the fact that the habit modification was a combined result of both

porosity and chemistry as stated before. The results reported here were obtained at least 8

times out of 10 experiments performed in case of Concanavalin A and 7 times out of 10

experiments performed in case of Catalase, which suggest higher degrees of reproducibility

as far as the crystallisation of macromolecules are concerned.

4. Conclusion

Controlling porosity and surface chemistry of heterogeneous nucleants are key considerations

for successful application in protein crystallisation process. Furthermore, comprehensive

characterisation of the nucleant properties is essential to outline a systematic correlation

between heterogeneous nucleants and the crystallising macromolecules. This work has been

focussed on using multiple characterisation techniques for rigorous analysis of the porosity

and surface chemistry of mesoporous templates. Here, we report on the methodology for

controlling the pore size distribution and average pore diameter of silica templates in the

range of 3-22nm by varying catalyst concentration, using sacrificial substrates and swelling

agents. This range of pore sizes is selected as they correlate to the hydrodynamic radius of

most of the common macromolecular proteins used in various processes. Utilising BET and

TEM measurements, we have obtained both qualitative and quantitative characterisation of

porosity of these materials. Upon functionalisation of these 3D nanotemplates, we have

confirmed the surface chemistry of the nucleants using multiple analysis techniques. While

contact angle and zeta potential are relative measurements providing valuable qualitative

information on the surface energy and surface charge of the templates, XPS is used as a

rigorous quantitative method to analyse the chemical composition of the surface.

Considerable importance is being placed on achieving an explicit knowledge on the surface

properties of the nucleants since the interactions that lead to heterogeneous nucleation of

proteins on these templates are significantly weighted on the interfacial properties. Influence

of heterogeneous nucleants on protein crystal habits is being observed for the first time by

using nucleants with varying surface chemistry. Catalase and concanavalin A are reported for

the first time to produce different crystal habits under the same crystallisation conditions of

pH, salt concentration, solvent and temperature with the application of 3D nanotemplates.

This emphasises the broad role that these templates can take in the future of

biochemical/biopharmaceutical crystallisation processes.

Acknowledgement

The authors acknowledge Prof. M. Jayne Lawrence and Dr. Laila Kudsiova, Kings College

London, for fruitful discussions and zeta potential measurements of templates prepared in this

study.

References

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List of Table Legends:

Table 1 Textural parameters and composition of 3D Nanotemplates prepared

Table 2 Experimental results of atomic composition (Carbon) and water contact angle of the 3D Nanotemplate surfaces functionalised with four different functional groups.

List of Tables:

Table 1 Textural parameters and composition of 3D Nanotemplates prepared

Variable Details Surface area (m2 g−1)

Total pore volume(cm3 g−1)

BJH model based average pore diameter

(nm)Catalyst Molar Ratio

TEOS:H2O:HNO3

Effect of Catalyst Concentration

1:12:0.03 827.3 0.221 1.93

1:12:0.3 803.2 0.414 2.19

1:12:1.2 594.8 0.591 3.94

Surfactant Type

Effect of Sacrificial Templates

Pluronic P123 612.1 0.679 5.31

Pluronic F127 547.2 0.814 10.21

Swelling Agent

Effect of Swelling Agent Pluronic F127(TMB)

496.6 0.896 14.92

Pluronic F127(Xylene)

299.3 1.063 22.32

Table 2 Experimental results of water contact angle and atomic composition (Carbon) of the 3D Nanotemplate surfaces functionalised with four different functional groups. The binding energy of the deconvoluted C1s spectra peaks are provided below the bond description.

Functional end-groups

Water contact Angle(deg)

XPS C1s spectra% Atomic

C-C C-Si C-N C-Cl C-C (aromati

c)

C-Si (aromati

c)285.0eV 284.3

eV285.8

eV286.8

eV284.7 eV 284.2 eV

Un-treated 11.30 ± 2.80

Amine 33.20 ± 4.10 8.90 8.85 8.82Chloropropyl 82.60 ± 3.50 10.91 11.21 11.13

Phenyl 109.10 ± 4.90 10.65 2.17

Dodecyl 128.60 ± 3.20 18.44 1.65

List of Figure Legends:

Figure 1 Pore size distributions of 3D nanotemplates produced with different silica precursor to catalyst molar ratios. Figure 2 Pore size distribution of monoliths produced with different sacrificial templates.

Figure 3 Transmission electron micrographs of nanoporous silica based 3D nanotemplate, (a) 5.51.5 nm (Scale: 20nm) and (b) 11.03.0 nm (Scale: 200nm).

Figure 4 Comparison of pore size distributions obtained with different swelling agents.

Figure 5 TEM of the sample prepared with Pluronic P123 as template and xylene as swelling agent, showing poor order of pore arrangement (Scale: 100nm).

Figure 6 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted C1s spectra. FWHM of 1.7eV for C1s spectra was employed.

Figure 7 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted Si2p spectra. FWHM of 2.2eV for Si2p spectra were employed.

Figure 8 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted O1s spectra. FWHM of 1.9eV for O1s spectra were employed.

Figure 9 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted N1s spectra. FWHM 2.2eV for N1s spectra were employed.

Figure 10 XP Spectra for 3D nanotemplate surface functionalised with chloropropyl functional end group showing deconvoluted Cl2p spectra. FWHM of 1.7eV for Cl2p spectra was employed.

Figure 11 Crystals of Concanavalin A on functionalised surfaces; (a) control (b) phenyl (c) dodecyl (d) chloro surface (scale: 200m)

Figure 12 Crystals of Catalase on functionalised surfaces; (a) control (b) phenyl (c) dodecyl (d) chloro surface (scale: 200m)

0 20 40 60 80 100 120 1400

0.1

0.2

0.3

0.4

0.5

0.6

0.71.20.03

Pore Diameter (Å)

Pore

Vol

ume

(cm

³/gÅ

)

Figure 1 Pore size distributions of 3D nanotemplates produced with different silica precursor to catalyst molar ratios.

0 50 100 150 200 250 300 350 400 450 5000

0.5

1

1.5

2

2.5

3

3.5

4

4.5Pluronic P123Pluronic F127

Pore Diameter (Å)

Pore

Vol

ume

(cm

³/g·Å

)

Figure 2 Pore size distribution of monoliths produced with different tri-block co-polymers.

TEOS:HNO3=1:1.2TEOS:HNO3=1:0.03

Figure 3 Transmission electron micrographs of nanoporous silica based 3D nanotemplate with average pore diameters of, (a) 5.5±1.5nm (Scale: 20nm) and (b) 16.0±3.0 nm (Scale: 100nm).

0 50 100 150 200 250 300 350 400 450 5000

1

2

3

4

5

6TMBXylenew/o Swelling Agent

Pore Diameter (Å)

Pore

Vol

ume

(cm

³/g Å

)

Figure 4 Comparison of pore size distributions obtained with different swelling agents.

(a) (b)

Figure 5 TEM of the sample prepared with Pluronic P123 as template and xylene as swelling agent, showing poor order of pore arrangement (Scale: 100nm).

278280282284286288290292294296

Amine Modified Surface C1sC1s C-CC1s C-SiC1s C-NC1s C-C-FEnvelope

Binding Energy (eV)

C1s

XP

Sign

al (a

.u.)

Figure 6 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted C1s spectra. FWHM of 1.7eV for C1s spectra was employed.

949698100102104106108110112

Amine Modified Surface Si2pSi2p -Si-OSi2p Si-CBackground

Binding Energy (eV)

Si2p

XP

Sign

al (a

.u.)

Figure 7 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted Si2p spectra. FWHM of 2.2eV for Si2p spectra were employed.

524526528530532534536538540542

Amine Modified Surface O1sO1s O-SiBackgroundEnvelope

Binding Energy (eV)

O1s

XP

Sign

al (a

.u.)

Figure 8 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted O1s spectra. FWHM of 1.9eV for O1s spectra were employed.

390392394396398400402404406408

Amine Modified Surface N1sN1s N-CEnvelopeBackground

Binding Energy (eV)

N1s

XP

Sign

al (a

.u)

Figure 9 XP Spectra for 3D nanotemplate surface functionalised with amine functional end group showing deconvoluted N1s spectra. FWHM 2.2eV for N1s spectra were employed.

192194196198200202204206208210

Chloro Modified Surfaces Cl2pCl2p Cl-CCl2p Cl-C1BackgroundEnvelope

Binding Energy (eV)

Cl2

p X

P Si

gnal

(a.u

.)

Figure 10 XP Spectra for 3D nanotemplate surface functionalised with chloropropyl functional end group showing deconvoluted Cl2p spectra. FWHMs of 1.7eV for Cl2p spectra was employed.

Cl2p3/2

Cl2p1/2

Figure 11 Crystallisation of Concanavalin A on functionalised surfaces; (a) control (b) phenyl (c) dodecyl (d) chloro surface (scale: 200m)

(a) (b)

(c) (d)

Figure 12 Crystallisation of Catalase on functionalised surfaces; (a) control (b) phenyl (c) dodecyl (d) chloro surface (scale: 200m)

(a) (b)

(c) (d)