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Bioinspired calcium phosphate coated mica sheets by vapour diffusion and its effects on Lysozyme assembly and crystallization Jaime Gómez-Morales, Cristobal Verdugo-Escamilla, and Jose Antonio Gavira Cryst. Growth Des., Just Accepted Manuscript • DOI: 10.1021/acs.cgd.6b00716 • Publication Date (Web): 15 Aug 2016 Downloaded from http://pubs.acs.org on August 17, 2016

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Crystal Growth & Design

Bioinspired calcium phosphate coated mica sheets by vapour diffusion and its effects on Lysozyme assembly and crystallization Jaime Gómez-Morales*, Cristóbal Verdugo-Escamilla, José Antonio Gavira Laboratorio de Estudios Cristalográficos, Instituto Andaluz de Ciencias de la Tierra (CSIC-UGR), Avda. de las Palmeras 4, 18100, Armilla (Granada), Spain.

Abstract We propose for the first time the vapour diffusion method to deposit bioinspired calcium phosphate films on mineral substrates, i.e. delaminated mica muscovite sheets, and to assess the capability of these films to affect the nucleation and growth of lysozyme crystals. Deposited calcium phosphate layers were composed of octacalcium phosphate (OCP) and apatite (Ap) nanocrystals, with increased amount of OCP at higher crystallization times. In the presence of polyacrylic acid (PAA) deposited layers were composed of amorphous calcium phosphate (ACP). Results of lysozyme crystallization showed that OCP/Ap-coated mica sheets slowed down the nucleation process without altering significantly the number of nucleated crystals per droplet respect to the uncoated control. ACP-coated mica sheets acted as an inhibitor, thus delaying the nucleation and reducing in addition the number of crystals. These results contrasted with the nucleation induction effect observed when calcium phosphate nanopowders were added to the crystallization drops. All coated-substrates promoted the formation of flat lysozyme crystals at the substrate-solution interface, and some of them retained its laminar morphology. Unexpectedly, the ACP coatings templated the 1 ACS Paragon Plus Environment


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2D assembly of lysozyme producing thin films. The observation of the lysozyme thin films was possible only in those ACP-coated supports prepared in presence of 0.015 and 0.02 wt% PAA.

Introduction Heterogeneous nucleation and growth of calcium phosphates salts (hereafter named as CaP) on metallic substrates (mainly Ti and its alloys) has been a subject of intensive research in the last two decades because of the significance of CaP- coated implants for load bearing applications in the orthopaedic and dental fields.1-12 Few studies, however, have been performed on the nucleation and growth of CaP on non-metallic substrates.1316

Besides its interest at a fundamental level, these studies may have either a biomedical

relevance in the preparation of implants for non-load bearing applications, or as a tool to understand heterogeneous nucleation in biological systems, or else, in the preparation of biomimetic functionalized surfaces with potential capability to induce the nucleation of protein crystals. In this field, positively and negatively charged surfaces (mica and polystyrene) ,17,18 imprinted polymers19 or porous nucleating agents20 have been proposed to promote the nucleation of some selected proteins. We have recently reported a new methodology, based on the vapour diffusion sitting drop micro-method (VDSDM), to precipitate nanocrystalline calcium phosphate apatite (Ap).21-23 Compared to stoichiometric hydroxyapatite [HA, Ca5(OH)(PO4)3], the obtained precipitate is Ca- and OH-deficient and is doped with CO32- ions. It can be represented by the general chemical formula Ca5-x(PO4)3-x(CO3)x(OH)1-x, with 0 ≤ x≤1. The method has been developed by using an innovative device, already employed for crystallization studies of calcium carbonate and biocrystallization, called crystallization mushroom.24,25 In this system the precipitation of CaP is driven by the decomposition of 2 ACS Paragon Plus Environment

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NH4HCO3 in CO2 and NH3 that diffuse through Ca-bearing microdroplets of around 40 µL. It was found that mixed solutions containing 50 mM Ca(CH3COO)2 plus 30mM (NH4)2HPO4 in the microdroplets and 3 mL of 40 mM NH4HCO3 in the gas generation chamber were the optimal to precipitate Ap nanocrystals.21 In this process the pH of the droplets started at around 5.6 and evolved till 8.5 in 6 hours. The nanocrystals were formed by solvent mediated phase transformation of octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP) to Ap. They showed carbonate ions in the crystal lattice, plate-like morphology, and a low crystallinity degree, closely resembling the inorganic phase of bone.

Contemporarily with this research, Nassif et al. also reported the

synthesis of biomimetic Ap by vapour diffusion using higher solution volumes (tens of millilitres) into a desiccator.26 The VDSDM revealed to be a powerful tool to synthesize Ap and to perform nucleation studies. Its use to induce the heterogeneous nucleation of CaP on a surface can be of great interest in order to develop a biomimetic route to coat any support with CaP. In this paper, we propose for the first time this technique to deposit CaP films on mineral substrates, i.e. mica muscovite sheets. Later on, the capability of these biomimetic films to control the nucleation and growth of protein crystals has been assessed in the crystallization mushroom using lysozyme (Lys) as model protein. Thin CaP layers composed of either OCP plus Ap or of amorphous calcium phosphate (ACP) have been produced. Interestingly, the ACP-coated samples behaved as an inhibitor of Lys crystallization and templated the 2D assembly of Lys molecules producing thin films.

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Materials and Methods Calcium phosphate deposition on mica sheets by vapour diffusion Delaminated mica sheets were dipped in a 0.1 M HNO3 solution for 2 h; then they were rinsed with deionized water and left to dry overnight into a desiccator. Stock solutions of Ca(CH3COO)2, (NH4)2HPO4 and NH4HCO3 were prepared using highpurity chemical reagents from Sigma Aldrich and ultrapure water (0.22mS, 25 ºC). Coating experiments were carried out in a crystallization mushroom (Triana Science & Technology, S.L.) at 20 ± 2ºC and 1 atm total pressure (Fig. 1). The diameter of the mushroom is 100 mm while the diameter of its central hole connecting the reservoir chamber with the crystallization chamber is 6 mm. In each experiment, 12 mica sheets were concentrically placed inside the upper chamber of this device. A solution droplet formed by mixing 20 µL of 50mM Ca(CH3COO)2 and 20 µL of 30mM (NH4)2HPO4 (Ca/P=5/3) was put on each sheet. Next, 3 mL of a 40mM NH4HCO3 solution were placed into the reservoir chamber. The glass cover and the upper chamber of the mushroom were sealed with silicon grease. The time of the experiments was 1, 7, 14 and 21 days. Additionally, the influence of polyacrylic acid (PAA) on the crystallization process was tested in two consecutive runs. In the first mushroom, the mica sheets (positions from 3 to 12) were covered by droplets prepared by mixing 10 µL of 50mM Ca(CH3COO)2, 10 µL 30mM (NH4)2HPO4 (Ca/P=5/3) and 20 µL of x w% PAA, with x=0.02 to 0.08. The sheets 1 and 2 were used for the blank experiment using pAA-free solutions.

In the second one, the only

difference was the value of x ranged from 0.005 to 0.02.


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Figure 1. Crystallization mushroom set up for deposition of calcium phosphate thin films on mica muscovite sheets by the sitting drop vapour diffusion method.

After closing and sealing the mushroom, the released gases NH3 and CO2 emerging from the underlined NH4HCO3 solution slowly diffuse to the upper chamber and dissolve into the solution droplets which cover the mica sheets, composed of complexed Ca-CH3COO+ plus H2PO4-/HPO42-, thus increasing its pH. This increase in pH provokes destabilization of Ca-CH3COO+ complexes and then the heterogeneous nucleation of calcium phosphate induced by the mica sheets. At the end of the experiments, the mushrooms were opened and the sheets were rinsed with deionized water and left to dry at ambient temperature or at 120 ºC for 1 day. Lysozyme crystallization on calcium phosphate-coated mica sheets First, several screenings of crystallization conditions of Lys by vapour diffusion were carried out using the mushroom (Figure S1 in Supporting Information). They were performed by varying the initial concentration of Lys between 5 and 25 mg/mL and the concentration of NaCl from 2 to 4 wt% in the droplet and from 4 to 8 wt% in the reservoir. The volume of the NaCl solution was 5 mL while the droplet volume was 4 µL. After the screenings, the conditions 25 mg/mL Lys, 3.5 wt% NaCl (in droplets) and 5 ACS Paragon Plus Environment


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7wt% NaCl (in the reservoir) were chosen as reference for the blank experiment. In these conditions, the average waiting time for crystallization was 2-3 days, and the number of crystals per droplet counted after 4 days, varied between 6 and 9. For the nucleation tests, selected CaP-coated mica substrates withdrawn from experiments at 1and 14 days as well as from experiments at 21 days in presence of PAA (0.005-0.02 M) were used to support the Lys crystallization droplets in 10 out of the 12 positions of the mushroom. The two other positions were occupied with uncoated mica and a blank droplet without mica. In a second set of experiments we set-up two mushrooms with only three droplets on mica, CaP-coated mica (the 14 days coating experiment) and the blank on silanized glass. Additionally, a third set was designed to include CaP powder to the protein-precipitant crystallizing drops. The mushroom was used to set-up 6 droplets: 2 blanks on silanized glass, 2 with 0.5 mg of nanocrystalline apatite (nanoAp) and 2 with 0.5 mg amorphous calcium phosphate (ACP) nanoparticles. In this set, glass cover slips were used as supports. Both nanoAp and ACP were prepared by the method of thermal decomplexing of Ca/citrate/phosphate/carbonate solutions.23At the end of the experiments the Lys crystals formed were stabilized by cross-linking with a 5 wt% glutaraldehyde solution for further manipulations. Characterization The CaP-coated mica sheets were characterized by optical microscopy (OM), X-ray powder diffraction (XRD), Raman spectroscopy and variable pressure scanning electron microscopy (VPSEM). Lys crystals were visually observed under OM and VPSEM. For OM analysis, either we employed a Leica MZ12 or an Olympus SZH10 connected to a digital camera (Olympus, C3040ZOOM). X-ray powder diffraction (XRD) patterns were collected using Cu Kα radiation (λ = 1.5418 Å) on a PANalytical X’Pert PRO


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diffractometer equipped with a PIXcel detector operating at 45 kV and 40 mA. For the incident and diffracted beams, automatic-variable anti-scatter slits with a constant irradiated length of 10 mm were used. The 2θ range was from 4º to 80º with a step size of 0.026º. For XRD characterization, the CaP coating was detached from mica by scratching at the surface. Raman spectroscopy measurements were performed with a JASCO NRS-5100 Micro-Raman spectrometer. A diode laser emitting at a wavelength of 532 nm provided the excitation line. A Peltier cooled charge-couple device (CCD) (1064 6 256 pixels) was used as detector. Spectrometer resolution is better than 1 cm1

/pixel at 100 cm-1 and excitation of 532 nm. The acquired spectra were linearly base

line corrected for clarity. Both the CaP-coated mica sheets and the cross-linked lysozyme crystals were inspected with a variable pressure Zeiss SUPRA40VP scanning electron microscope (VPSEM). The instrument is provided of a large X-Max 50 mm area detector for X-ray dispersive energy (EDX) analysis. EDX values of Ca and P for each CaP-coated sample have been averaged from 7-10 different measurements. Additionally, we have measured the wetting angles of deionized water droplets deposited on silanized glass, mica muscovite sheets and CaP-coated mica. Wetting angles were determined using the sessile-drop method at room temperature18. 5 µL of deionized water were dropped onto the different surfaces. The droplet was left undisturbed for about 1 min and its shape was then recorded with a digital camera. Measurements were repeated five times.

Results a) Calcium phosphate coatings on mica sheets Figure 2 shows the characteristic features of CaP layers deposited at 1, 7, 14 and 21 days of vapour diffusion at ambient temperature. A general view of these layers and of 7 ACS Paragon Plus Environment


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the heated specimens at 120 oC is shown in Figure S2 in Supporting Information. It is worthwhile to emphasize the production of CaP coatings with a high degree of homogeneity in the zones of mica covered by the droplets, with small cracks and imperfections, mainly attributable to post-treatment manipulations. At 1 day, the deposited layers of about 1-2 µm thickness were composed of needle-like apatite crystals of 100-200 nm length and small platelets of octacalcium phosphate (OCP). The average Ca/P ratio determined by EDX was 1.78 ± 0.06, higher than that of the stoichiometric hydroxyapatite (1.67). The thermal treatment at 120 ºC increased the crystallinity of the layers without changing the crystal morphology. The average Ca/P ratio of the thermally treated products was 1.62 ± 0.02. After 7 days of vapour diffusion, the deposited layers of about 2-3µm thickness were composed of Ap nanocrystals and a higher amount of long OCP blades of about 11.5 µm in length and 50 -100 nm width (see insert of Figure 2b for further details). The OCP crystals show wide (100) faces elongated along the c-axis and are bordered by {011} and {010} forms. The average Ca/P ratio decreases to 1.52 ± 0.03, closer to that of OCP (1.33) than the former sample. The scratch carried out on the layer surface (Figure 3) produced a plastic deformation instead of the typical fracture causing layer delamination, which indicates that they are strongly attached to the mica substrate. At 120 ºC, the Ap needles and OCP blades coexist in the layers.


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Figure 2. CaP layers deposited at ambient temperature by sitting drop vapour diffusion technique on mica sheets at 1 day (a), 7 days (b), 14 days (c) and 21 days (d). Insert in image (b) shows the characteristic morphology of OCP blade crystals displaying the (100) faces elongated along the c-axis and bordered by {011} and {010} forms. Mixtures of OCP plus Ap nanocrystals populated the layers.



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Figure 3. Details of cracks and scratches performed in CaP layers deposited at ambient temperature by sitting drop vapour diffusion method on mica sheets at 1 day (a), 7 days (b, c), and 21 days (d).

Samples obtained after 14 days were mainly composed of long prismatic OCP plates of about 2 µm in average length and 100-150 nm width, with little amount of apatite nanocrystals. The average Ca/P ratio of the samples is 1.34 ± 0.04, similar to that of OCP. The thermal treatment of these layers at 120 ºC produced the widening of the crystals, similarly to the observed in samples obtained at 7 days. The picture is similar when samples were removed after 21 days of vapour diffusion. Once again, at ambient temperature and at 120 ºC a higher amount of long OCP crystals populates the layers while nanocrystalline apatite appears as the minor phase. The scratch of the layers removes material producing a groove but does not yield the delamination of the sample. The XRD diagrams of the samples prepared at the different times (Figure 4) show the distinguishing reflections of either the support (+), the apatite (#) (PDF 01-1008) 10 ACS Paragon Plus Environment

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and the octacalcium phosphate phase (*) (OCP, PDF 44-0778). A detailed assignment of peaks to these two CaP phases is shown in Figure S3 of Supporting Information. For this purpose, the acquisition time of the diffractograms was 40 h. The main XRD reflections of the Ap appear at 2θ 25.87º (plane 0002), 31.77º, 32.19º and 32.90º (planes 21-31, 11-22 and 30-30) and other minor peaks at 39.81º and 53.14º which correspond to reflections 31-40 and 0004 respectively27,28. The distinguishing XRD reflections of OCP appear at 4.74º (100), 9.74º (010), and 23.7º (311). Other peaks of OCP emerge in the same region of the Ap, being difficult to distinguish both phases.

Figure 4. XRD diagrams of the layers at 1 day (blue), 7 days (brown), 14 days (orange) and 21 days (green). Apatite peaks are marked by #, OCP peaks by * and mica muscovite peaks by +. The last peaks are labelled only in the 21 days diagram to avoid overlapping with the other diffractograms. Note that XRD diagram of the deposit at 2 weeks has been recorded under grazing incidence of 1º.



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Using these XRD diagrams, we have determined the ratios between the area peak at 4.74º (OCP) and that of the wide reflection located between 31º and 33º (where peaks of OCP and Ap are overlapped). The results, 0.32 (1 day), 0.50 (7 days) and 0.78 (21 days) indicate the increase in the mass proportion of OCP respect to Ap when the crystallization time increases.

Figure 5. Left. Raman spectra of the layers deposited at 1 (orange), 7 (green), 14 (brown), 21 days (yellow) and mica (blue). Right. Raman spectra of calcium phosphate layers deposited at 21 days and 21 days in presence of 0.005 wt% PAA. The insert shows the XRD diagram of the amorphous calcium phosphate layer deposited in presence of 0.005 wt% PAA.

The presence of OCP is also revealed by the bands at 958 cm-1 and 966 cm-1 (shoulder) and the broad band at around 1010 cm-1 in the Raman spectra (Figure 5, left), characteristics of the P-O symmetric stretching modes of PO4 (υsPO4) and HPO4 (υsHPO4) in the OCP phase23. In the presence of PAA, even at the lowest concentration used (0.005 wt%), the layers are composed of ACP. The amorphous nature of the deposit is confirmed by both the P-O symmetric stretching mode of ACP, υs(PO4), at 954 cm-1 in the Raman spectra and by X-ray diffraction, evidenced by the absence of the

characteristics reflections of CaP (insert of Figure 5, right). The as-prepared ACP layers 12 ACS Paragon Plus Environment

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at PAA concentrations >0.02 wt% are highly homogeneous in thickness in spite of they are fragmented and poorly attached (Figure 6 c and d).

Figure 6. Amorphous calcium phosphate layers deposited at ambient temperature by SDVD technique on mica substrates at 21 days in presence of 0.0 w/wt% PAA (a), 0.02 w/wt% PAA (b), 0.04 w/wt% PAA (c) and 0.08 w/wt% PAA (d). Inserts show the morphology of the particles composing the layers. Note that in presence of PAA the layers are composed of nanosized spheroidal ACP particles.

b) The influence of CaP-coated mica on the crystallization of lysozyme Table 1 summarizes the main results of the different sets of lysozyme crystallizations. In it, tw (days) is the average waiting time for crystallization, i.e. the elapsed time from the onset of the experiment up to the appearance of the first crystals



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(observed daily under an optical microscope at magnification 4×) and Ncrystals is the average number of crystals per droplet. The two last rows show the initial and final features of these crystals. For the sake of simplicity, the morphologies have been classified as bulky, i.e. standard tetragonal, point group 422, crystal shape in which the typical bi-pyramidal {101} and prismatic {110} faces are clearly observed, or laminar, i.e with direction [001] parallel to the surface plane. Results in the first set of experiments show that in the blank droplet supported on the silanized glass of the mushroom the tw was of 2-3 days and the experiment yielded bulky crystals. Similar tw, 2-3 days, was observed in droplets containing uncoated mica sheets. In this case the crystals observed initially displayed laminar morphology, most probably corresponding to the {110} faces. In droplets containing OCP/Ap-mica the tw were longer, 4-5 days, and though crystals grew to a bulky shape, some of them retained their initial laminar shape. In the remaining droplets containing ACP coatings the number of crystals was the lowest, 2-3, evidencing the highest inhibition effect of these films. Crystals were initially laminar and some of them retained its morphology, as in the former cases. Notably, the experiments using the ACP-mica supports obtained in presence of 0.02M PAA, when inspected under VPSEM at 20 and 5 Kv, unambiguously revealed the formation of Lys thin films in addition to the laminar and bulky crystals (Figure 7A-F). This thin Lys film, of 200-400 nm thickness, is very difficult to observe and has been only found on ACP-mica deposited in presence of 0.015 and 0.020 M PAA but not in the rest of ACP-mica experiments.


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Table 1. Influence of CaP-mica layers on the crystallization of tetragonal lysozyme

Droplet

CaP layer composition

tw (days)

Ncrystals

Initial morphology

Final morphology

1st set

1

OCP/Ap 1 day, 120ºC

4-5

13

Laminar

Bulky

2,3

OCP/Ap 14 days,120ºC

4-5

12

Laminar

Bulky

4

OCP/Ap 1 days, Tamb

4-5

8

Laminar

Bulky + laminar

5,6

OCP/Ap 14 days, Tamb

4-5

8

Laminar

Bulky + laminar

7

ACP (0.005 M PAA)

4-6

2

Laminar

Bulky

8

ACP (0.010 M PAA)

4-5

2

Laminar

Bulky

9

ACP (0.015 M PAA)

4-5

3

Laminar

Bulky +laminar

10

ACP (0.020 M PAA)

4-5

2

Laminar

Bulky +laminar

11

Blank (mica muscovite)

2-3

9

Laminar

Bulky

12

Blank (glass)

2-3

8

Bulky

Bulky

2nd set

1

Blank (mica muscovite)

1-2

10

Laminar

Bulky

2

OCP/Ap 14 days Tamb

>3

9

Laminar

Bulky

3

Blank (glass)

1-2

4

Bulky

Bulky

3rd set

1

NanoAp (0.5 mg)

1

>50

Bulky

Bulky

2

ACP (0.5 mg)

2

25- 30

Bulky + laminar

Bulky +laminar

3

Blank (cover slip )

5

7, 6

Bulky

Bulky



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Figure 7. VPSEM micrographs of Lys crystals grown on ACP-coated mica sheets obtained in presence of 0.020 wt % PAA. (A, B) Bulky crystals; (D, F, H) lysozyme thin films and (C, E, G) agglomerated crystals formed on these films. Micrographs A, B, C, E and G were carried out at 20 Kv while D, F and H were taken at 5 Kv. Note that Lys films are fragmented due to manipulation.


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a)

b)

Figure 8. Elemental % mol composition of C, O, N, Ca and P determined by X-ray dispersive energy (EDX) analysis along random lines drawn on two different Lys-ACP-mica (0.02%wt PAA) regions. Micrograph (a) recorded at 5V: from left to right shows a flat Lys crystal, a Lys thin film, ACP and Lys thin film. Micrograph (b) recorded at 20 KV shows the Lys thin film formed on ACP and a laminar Lys crystal.

Figure 8 shows the EDX analysis along the random lines drawn on two different regions of the Lys-ACP-mica (0.02%wt PAA) sample. From left to right, EDX analysis in figure 8a reveals, as expected, a segment rich in C and N, poor in P, Ca and O



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corresponding to a Lys crystal, a segment showing intermediate concentration in C and N (thin Lys film), the segment rich in Ca and P, poor in C and N, corresponding to the ACP layer and the one with intermediate concentration in C and N (Lys film). The elemental composition of C, N, Ca and P along the line in figure 8 b corresponds to the Lys thin film formed on ACP and the laminar Lys crystal in the middle. In the second set of crystallization trials, the tw was 1-2 days for the experiments blank and mica while in the OCP/Ap-mica the Lys crystals appeared after 3 days. In all experiments of this set the final observed Lys crystals were bulky and almost isometric. There were no evidences of Lys thin films formed on these experiments. In the third set, after 2 days, we found bulky Lys crystals on the nanoAp- and nanoACP-bearing droplets while nucleation of Lys on the glass cover slips was achieved after 5 days of vapour diffusion. Both CaP nanopowders promoted the nucleation of Lys. All crystals showed the standard bulky Lys shape with no evidences of laminar or thin film formation neither on nanoAp nor on ACP powders. Discussion a) Calcium phosphate coatings on mica sheets This work shows the presence of a mica muscovite sheet in contact with microdroplets yields the formation of thin coatings composed of OCP and Ap nanocrystals with increased proportion of OCP as time increases. Additionally, when using PAA as additive the coatings are composed of ACP nanoparticles. This trend is different to that observed in previous experiments using silica gel29 or those carried out by vapour diffusion method in absence of a substrate.21,23,30 In the last references the formation of ACP nanoparticles, just after mixing the reagent solutions, was followed by the appearance of OCP after 1 day and then by Ap nanocrystals after 1 week of 18 ACS Paragon Plus Environment

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vapour diffusion. The time resolved experiment, monitored by in situ Raman microspectroscopy23, revealed that ACP transformed first to dicalcium phosphate dihydrate (DCPD, brushite) and then to OCP plus Ap. The lifetime of DCPD strongly depended on the concentration of the NH4HCO3 solutions and thus on the pH increase rate. The pathway for the phase transformation from ACP to DCPD and then to OCP was a dissolution–reprecipitation mechanism. Additionally, OCP acted as temporal template for the heterogeneous nucleation and growth of Ap nanocrystals. Finally OCP dissolved. This pathway was altered in presence of aminoacids30. The ACP transformed to dicalcium phosphate dihydrate (DCPD; brushite) in presence of L-aspartic acid (Lasp, iep = 2.77) or to OCP plus Ap nanocrystals in the presence of L-alanine (L-ala, iep = 6.00). The alteration was explained because of the influence of these aminoacids on the pH evolution in the droplet and on the nature and strength of the interactions of the major charged species of the amino acids with the surface lattice ions of the DCPD or OCP phases, which acted as precursors of the Ap phase. Base on the findings of the present research we propose that mica triggers the heterogeneous nucleation of ACP. Later on, ACP transforms to OCP by a dissolutionreprecipitation process at the substrate-droplet interface and then OCP templates the formation of Ap nanocrystals, but does not dissolve, in contrast to the previously published pathway.30 Once the OCP phase formed, it was stabilized, most likely by interacting the rough surface through its large {100} face. Note that there is no preferential orientation of the OCP crystals along the c-axis with respect to the mica sheets. The stabilization at the substrate-solution interface avoids OCP dissolution and then, as time increases, new OCP crystals are formed as well as few Ap nanocrystals by the reported mechanism in which OCP acted as a template. The presence of OCP and Ap nanocrystals is responsible for the strength of the layer attachment to the substrate as 19 ACS Paragon Plus Environment


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well as of its plastic deformation. Only in the presence of PAA, the ACP to OCP transformation is fully inhibited and the strength of the layer attachment to the surface of mica was worse than in layers composed of OCP plus Ap. The first step in the whole phase transformation pathway is the heterogeneous nucleation of ACP on mica. Heterogeneous nucleation is normally explained in terms of contact angle values based on the wetting of the solid surface by the embryos. In addition, in this case the Lewis acid–base surface site31 and the surface topography,32 should play a crucial role by providing an additional thermodynamic reduction in the free energy barrier to nucleation. The templated transformation of OCP to Ap can be related to the remarkable structural similarities displayed by both phases. Indeed, OCP crystallizes in the triclinic system, spatial group P-1. The unit cell of OCP consists of apatitic layers where Ca2+ and PO43- ions occupy the same positions as in the hexagonal structure of the apatite, and of hydrated layers, where Ca2+ and PO43- ions are more widely spaced, due to the presence of the interdispersed structural water molecules.33,34Apatitic layers alternate with hydrated layers parallel to the (100) face. Morphologically, OCP crystallizes as {100} blades of triclinic pinacoidal symmetry, elongated along the a-axis and bordered by the forms {010}, {001} and {011}33,34 (see figure 3.a). It is generally accepted that, in aqueous solution, the hydrated layer of the (100) face is the most likely exposed to solution. Thus, we assume this hydrated layer is the most likely involved in the born of the new apatite phase at the OCP-solution interface.


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b) The influence of CaP-coated mica on the crystallization and assembly of lysozyme The importance of heterogeneous nucleation of CaP on mineral substrates is limited to not only the biomineralization and medical fields. The preparation of bioinspired CaP-coated mica sheets responds to the renewed interest on using nucleating substrates for protein crystallization.19,35 In the present work, as an application of the bioinspired CaP-coated mica sheets prepared by vapour diffusion, we have tested its influence on the nucleation of Lys. It is worth noting the delay in the waiting time for crystallization with respect to tw observed in the blank experiments (without and with mica) and the different trend shown after adding synthesized Ap and ACP nanopowders. Contrarily to the effect observed with the surfaces, the added powders promote the nucleation, reducing tw and increasing suddenly Ncrystals. In addition, the formation of Lys crystals with “bulky” or “laminar and bulky” morphologies, starting from initially flat morphologies in many of the droplets, indicates the big influence of the CaP layer on the Lys features. In this context, the finding of the continuous thin film only on ACP layers is worth to be highlighted. To explain these results we assume that Lys crystals form either by heterogeneous nucleation or by homogeneous nucleation followed by adsorption to the substrate, as was already reported for calcium carbonate.36 The competition between the clustering of Lys molecules in the bulk droplets and the adsorption on the substrates with different wettability can help us to discuss the observed results. The wetting angle of the water droplets decreases from 46º ± 1o in silanized glass to 14º ± 1o in mica and to practically 0º in OCP/Ap-coated mica and ACP-coated mica (Figure S4) indicating the better wettability of the coated substrates. The decrease of the wetting angle is directly related to an increase of hydrophilicity and therefore a higher tendency to protein adsorption, 21 ACS Paragon Plus Environment


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reducing the nominal supersaturation with respect to the droplets of uncoated supports. The phenomenon is enhanced when decreasing the particle size as in the ACP coatings. The lower the particle size the bigger is the specific surface area of the substrate and so, the amount of adsorbed Lys molecules. Consequently, the availability of molecules to cluster and form nuclei is reduced, increasing tw. The 2D surface nuclei so formed tend to enlarge either three-dimensionally or bi-dimensionally if the remaining supersaturation after nucleation is enough to sustain their growth. In the experiments using ACP-mica (0.015 M, 0.020 MPAA), and probably in the rest of ACP-mica supports, although it was not detected, the Lys adsorption is so intense that Lys molecules self-assemble forming a continuous Lys thin film. On the other hand, when CaP is included as a suspension of particles, either as nanoAp or as ACP, the tw is reduced and the Ncrystals increases, when compare with drops free of particles. In this case, CaP acts as standard nucleant inducing the heterogeneous nucleation of Lys. In these experiments, bulk nucleation followed by adhesion to the surface of glass cover slips occurred, and Lys nuclei grew threedimensionally. Conclusions We report a new methodology, based on the vapour diffusion sitting drop method, to deposit thin CaP layers on mineral supports, i.e mica muscovite sheets. This method yields the formation of thin coatings composed of OCP plus Ap nanocrystals with increased amount of OCP as time increases. Additionally, when using PAA as additive the films are composed of ACP nanoparticles. Results of Lys crystallization showed that OCP/Ap-coated mica sheets slowed down the nucleation process without altering significantly the number of crystals per droplet respect to the uncoated control.


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ACP-coated mica sheets acted as an inhibitor, delaying the nucleation and reducing the number of crystals. In all coated-substrates, flat lysozyme crystals formed at the substrate-solution interface, and some of them retained its laminar morphology. Unexpectedly, the ACP coatings templated the 2D assembly of Lys molecules producing thin films. The observation of the Lys thin films was possible only in those ACP supports prepared in presence of 0.015 and 0.02 wt% PAA. Acknowledgements We greatly acknowledge the project Biomin-nanoapatite MAT2014-60533-R supported by Spanish MINECO and co-funded by FEDER. References 1

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Table of contents

Synopsis Calcium phosphate coatings on mica sheets have been deposited for the first time using the vapour diffusion sitting drop method.

The coatings composed of

octacalcium phosphate plus nanoapatite slowed down the nucleation of lysozyme without altering the number of nucleated crystals. Those coatings composed of amorphous calcium phosphate nanoparticles inhibited the nucleation of lysozyme crystals and some of them templated the 2D assembly of lysozyme molecules producing thin films.


Bioinspired Calcium Phosphate Coated Mica ... - ACS Publications

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