Crystal Assembly: The Application of High Affinity Ligands and Habit Modification N.
Blagden*,†
and B. R.
Heywood‡
School of Pharmacy, University of Bradford, West Yorkshire BD7 1DP United Kingdom, and Department of Chemistry, Keele University, Keele, Staffordshire, ST5 5BG United Kingdom
CRYSTAL GROWTH & DESIGN 2003 VOL. 3, NO. 2 167-173
Received July 3, 2002
ABSTRACT: Streptavidin-biotin was found to induce the aggregation of micrometer-sized calcite particles, and the optimum ligand-receptor surface tagging condition was found when monolayer coverage of the sample by the adsorbed ligand-receptor components was reduced to 80% coverage. The role of crystallography in the agglomeration process induced by the presence of surface-adsorbed ligand-receptor was also examined. This was achieved by using calcite exhibiting three different crystal habits: rhombohedral, hexagonal spindles, and triangular plates. The latter two habits express faces generated by anionic templates, and the topology of agglomerates suggests that the carboxyl group of the biotin and the streptavidin interacting with these surfaces generated a local variation in crystal packing. This was accounted for by simply relating the preferential interaction between the modified surface of the crystal and the high-affinity ligand complex on the functionality of additives used to template surfaces during crystal growth. Introduction Processing and product formulation involving dispersions and colloidal fluids are widespread in a variety of contemporary materials, e.g., ceramics, paints, inks, semiconductors, optical activity, and drug delivery. Central to the application of dispersions and complex colloidal fluids is the manipulation of floc formation, the density of aggregation, and the overall geometry and shear behavior of the system. The enhancement of bulk properties over those of particulate species of which the dispersion is composed is usually achieved by improving the overall anisotropy of the aggregating system.1 To date, the particle interactions involved in such systems rely solely on the manipulation of the isotropic interaction whether through electrostatics, as described by Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, or through surface-adsorbed surfactants or polymers, which generate a steric barrier to particle aggregation.2,3 However, there are known examples where anisotropic aggregation can be the result of crystallographic factors, where coagulation arises from dissimilar double layers,4 a classic example being for the clay, kaolin, where the profile of surface charge varies as a consequence of the crystal habit of the clay particles. In this case, the underlying crystallography and the expressed habit of the particle determine the aggregation topology. Other examples have been reported5-7 for silver iodide, hematite, and ferihydrite where the underlying morphology results in aggregation. Crystallographic considerations such as those found for kaolin have been employed to manipulate the aggregation of barium sulfate8 and zinc oxide.9 The underlying requirement is that the habit of the target particles is tuneable to a polar morphology. Molecular recognition and selfassembly strategies are employed to design molecular templates, which are able to manipulate the nucleation † ‡
University of Bradford. Keele University.
and growth of crystals. This enables specific interactions between a molecular template and a set of target crystal faces to be used to control the crystal size and habit of both inorganic and molecular crystals. Consequently, crystal habit modification can be used to tailor crystalline particle with specific surface stereochemistry and surface charge anisotropy.10 The literature contains numerous references to molecular complexes of biological origin (referred to as high-affinity ligand or ligand-receptor complex; the latter is used throughout this work, and two components are assumed), the most common being streptavidin-Dbiotin, lectin’sssugars and antibodiessantigens. Such a body of work also indicates that the complexation of such high-affinity ligands remains intact, even when one conjugate is supported on a host surface.11-14 The interest in such surface-supported reagents relates to enzyme separation, immunoassay, and biocatalytic conversion applications.15 Consequently, the kinetics of ligand-receptor complex formation at solid-liquid interfaces is generally well-understood.16 Parallel to this work is the analysis of surfaces containing these ligandreceptor macromolecules, where it has been shown that a specific binding interaction between sorbed macromolecule species and substrate occurs. The stereochemistry inherent to the geometry of the surface is reflected in the self-assembly of the bound component of the ligand-receptor complex on the substrate surface.17,18 This is also widely reported to occur for surfactants,19 lipids,20 and proteins bound to a variety of organic and inorganic substrates.21 The complexation behavior of surface-bound ligand receptors has been used to mediate the aggregation of nanoparticles. It has been shown that both colloidal superparamagnetic iron oxide22 and metallic particles of gold23 can be assembled with the streptavidin-biotin ligand-receptor complex. The kinetics of the aggregation process involving nanoparticles and controlled ligand-receptor addition and adsorption has been recently analyzed,24 and the potential of this approach to particle assembly has been discussed.25
10.1021/cg020024c CCC: $25.00 © 2003 American Chemical Society Published on Web 01/22/2003
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Figure 1. Envisaged scheme for ligand complex-mediated crystal aggregation. (a) Surface adsorbed with one component of ligand complex; (b) tagging of surface with second component of ligand complex and conjugate formation; and (c) ligand-stabilized binding via adsorbed surfaces.
Particle Aggregation through Crystal Habit and Surface-Bound Ligands. An ongoing issue within this area is to induce anisotropic interactions between particles. One possible avenue in which to introduce anisotropy interaction between particles is to use surfacebound/-adsorbed ligand receptors in conjunction with habit-modified particles. This approach builds upon particle aggregation strategy based on purely electrostatic stabilization through electric double layers and particles with polar morphologies or steric stabilization through polymers or surfactants, and the sequential addition of ligand-receptor components, which adsorb to the surface and mediate aggregation on ligandreceptor complex formation. Habit-modified crystals are employed to enhance the adsorption of the high-affinity ligands, thus adjusting the strength of binding. A scheme of the envisaged process is given in Figure 1. A suspension of habit-modified crystals is initially treated with the first component of ligand-receptor complex at a preselected concentration; this leads to the adsorption of the first component of the ligand-receptor complex onto the surface of crystals after a period of incubation (stage A of Figure 1). This adsorbed crystal suspension is then washed and resuspended and then treated with the second component of the ligand receptor at a preselected concentration (stage B of Figure 1). The resulting effect of the second component adsorption onto the surface of the suspended crystals and the subsequent ligand-receptor complex formation between the surface-bound ligand-receptor components on crystal aggregation and the topology is then monitored. In the work reported in this paper, the roles of both habit and ligand-receptor addition order and conditions were investigated. Model System. The system chosen to examine whether an anisotropic assembly could be achieved using surface-bound ligand-receptor complex formation in conjunction with crystal habit variation consisted of calcite as the microcrystalline solid supporting the ligand-receptor complex D-biotin {B}-streptavidin {S}. The D-biotin-streptavidin ligand-receptor complex was chosen since the kinetics and the high affinity of binding (K ) 1015) have previously been shown to be unaffected by being adsorbed to a substrate surface.26,27 Literature X-ray studies28,29 indicate that the high-affinity ligand interaction involves hydrogen bonding between the
Figure 2. Schematic of the streptavidin-biotin interaction. (a) Guest host geometry of 1:1 complexation and (b) nature of binding site.
carboxyl group of aspartic acids moieties on {S} and the urido group in {B} with four binding sites present within streptavidin (a schematic of the adduct geometry is shown in Figure 2). These kinetic studies have also indicated that both components of the ligand-receptor complex would have available carboxylate functionality capable of binding with a mineral surface once the complex between components was achieved. Calcite was chosen as the inorganic phase because the crystal growth is well-understood and routine protocols for habit modification of the calcite crystals are available. Thus, the choice of alternative morphologies would enable the role of crystal habit in receptor ligandmediated aggregation of inorganic particles to be examined, as different surface specific faces for complex formation would be present. The three morphologies used during the course of this work were as follows: (i) rhombohedral calcite, expressing (104) faces only as the control for this study; (ii) hexagonal spindle calcite, expressing both (104) and {110} faces. This morphology expresses {110} faces that contain both calcium and carbonate ions, which are stereochemical specific to carboxylate ions and would be specific to carboxylate-containing sorbates such as D-biotin and streptavidin. Consequently, the effect of preferential association of face due to surface stereo-
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chemistry would be examined. (iii) Triangular plate calcite expresses both (104) and {001} faces. This triangular plate morphology is polar across the {001} faces and consists of oppositely charged (001) faces composed of either a calcium rich face or a carbonate rich face, respectively. Consequently, the calcium rich face would be highly specific stereochemically for carboxylate-containing sorbates such as D-biotin and streptavidin; so in this case, assembly of anisotropically charged particles by the ligand complex would be examined. Experimental Section Because of the combinatorial nature of the procedures used, only generic methods will be given. Combinations of experiments were undertaken to examine the behavior of micrometersized rhombohedral calcite as the order of adsorption and concentration of ligand receptor was varied. The extent of aggregation was determined from optical micrographs of untreated and ligand receptor-adsorbed crystal suspensions. Factors affecting the level of aggregation were also examined including the order of addition, electrolytes, incubation temperature, and application of shear. The aggregation behavior of untreated and one component-adsorbed rhombohedral calcite was used as the benchmark controls for this study. Studies of the effect of habit on aggregation induced by the adsorbed ligand-receptor were also undertaken for both triangular and hexagonal morphologies of calcite and compared with that of the rhomohedral calcite. The evidence for an effect from the crystal habit on the geometry aggregation was generated from preferential association of faces based solely on visual inspection of face to face contacts of dried down settled particle samples under scanning electron microscopy (SEM) examination. Calcium Carbonate Crystallization and Morphologies. Calcite expressing three distinct morphologies was used. The following notation is to denote calcite type, C(n) where n ) r for rhombohedral, n ) h for hexagonal spindle, and n ) t for triangular plates. Rhombohedral calcite, C(r), expressing all {104} faces, was grown using the calcium hydrocarbonate solution procedure described by Kitano.30 Calcite expressing a hexagonal spindle form C(h), consisting of {104} and {110} faces, was obtained from a malonic acid-doped calcium hydrocarbonate solution,31 again the Kitano procedure was used to generate the calcite. The triangular plate modification of calcite consisting C(t), of {104} and {001} faces, was obtained by a modified Kitano method involving n-eicosyl sulfate monolayer at the solution-air interface and with the addition of lithium chloride to calcium hydrocarbonate solution subphase.7 Samples were washed three times in distilled and deionized water (3 × 1 cm3), with 12 h between each solvent exchange, to ensure minimal contamination of the surfaces of the preformed crystals. General Labeling Procedures. Four distinct sets of ligand-receptor adsorption sequences were undertaken on the three types of preformed calcite suspensions at one preset ligand-receptor component concentration. The preset conditions used were either 10-5 M streptavidin (Fluka) or 10-3 M biotin (Fluka) for the adsorption of receptor-ligand complex components onto the calcite. These concentrations were determined by adsorption isotherm work and correspond to concentrations required to achieve monolayer coverage of 1 mg of calcite suspended in 1 mL. The calcite suspension was either adsorbed with one component only onto the calcite, streptavidin; {C(n)S} or D-biotin only {C(n)B}; or both components were adsorbed sequentially onto the calcite, in the sequence streptavidin and then D-biotin {C(n)SB} or D-biotin and then streptavidin {C(n)BS}. For each type of adsorption sequence, 1 mg of preformed calcite was suspended in water (0.99 cm3) with 0.01 cm3 of stock 10-5 M streptavidin or 10-3 M biotin solution added as required. The sample was then incubated for 24 h at 4 °C. All samples were collected by
Crystal Growth & Design, Vol. 3, No. 2, 2003 169 sedimentation, washed in water (1 cm3), and resuspended in deionized water (either 0.25 cm3 prior to aggregation analysis or water (1 cm3) prior to further ligand-receptor component adsorption). For ease of convention, all calcite label experiments were denoted by the molarity of the stock solution (0.01 cm3) used. For the rhomobohedral calcite, a concentration series of ligand-receptor labeling was also carried out. The streptavidin concentration series used was between 10-4 and 10-8 M (typically 1/10 dilution steps were used), and the biotin concentration series was between 10-2 and 10-10 M (typically 1/10 dilution steps were used). For all of the sequential labeling work in the concentration series study, the D-biotin solution was 100 times more concentrated than the corresponding streptavidin solution used to label the sample to ensure ligand-receptor formation. Procedures for Aggregation Analysis. The extent of aggregation was monitored by calculating the aggregation index of the respective crystal suspension. The aggregation index was defined as [〈Nagg〉 × %Nagg/100], where 〈Nagg〉 is the average number of crystals in an aggregate and %Nagg is the level of aggregation, evaluated by determining the percentage of sample that had formed aggregates of two or more crystals. Aliquots (0.01 cm3) of crystal supension were examined visually by recording micrographs. These micrographs were used to determine the 〈Nagg〉 and %Nagg of a suspension sample and to calculate the respective aggregation index. To obtain a representative sample, up to four micrographs were taken for each aliquot of suspension examined, and 3-4 aliquots were examined in this way per suspension. In all cases, a sample consisted of more than 100 crystals. This analysis was taken as representation of the settled agglomerated particles, with the aggregation behavior of the unlabeled calcite taken as the control. Throughout this study, the aggregation behavior of unlabeled calcite was taken as the reference for aggregation in all of the single and fully labeled systems. SEM analyses of dried down aggregates obtained by the sampling procedure for optical analysis was employed to examine the detail of the face-to-face contacts as labeling conditions were varied for the habit-modified crystal studies.
Results Rhombohedral Calcite Studies. Micrographs illustrating the typical response to ligand-receptor addition are given in Figure 3, and the overall aggregation index profile for the concentration series undertaken is given in Figure 4. In Figure 3, a micrograph of an untreated sample is shown in Figure 3a and the {C(r)SB} sample, where both components of the ligand receptor are adsorbed, is shown in Figure 3b. (a) Outcome of Both Components of LigandReceptor Complex being Adsorbed ({C(r)SB} and {C(r)BS}). On addition of both components of the ligand-receptor complex, both of the {C(r)SB} and the {C(r)BS} concentration series micrographs gave an aggregation index profile, which indicated that a significant level of aggregation occurred for these samples. For the {C(r)SB} sample, typically more than 78% of the sample contains an average of seven more crystals in agglomerate at an optimum ligand concentration, as indicated by the profile of aggregation index vs addition concentration in Figure 4a. This corresponds to an aggregation index of six for the {C(r)BS} sample, and corresponding values for untreated calcite are typically 43% of the sample containing an average of three crystals and a corresponding index aggregation of 1.29. The optimum condition for aggregation of the rhombohedral calcite was found to occur for a {C(r)SB} addition order when the conjugates concentration, as indicted by
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Figure 3. Micrographs of rhomohedral calcite samples: (a) untreated C(r) and (b) both components of the ligand receptor adsorbed onto calcite, {C(r)SB}; note bar 200 µm.
the previous isotherm studies,32 was in or near the monolayer region. This corresponds to 1 mL of 1 mg per cm3 suspension of C(r) adsorbed with either {S} using 0.01 cm3 of the 10-5 to 10-6 M stock solutions (at this level of addition, the isotherm studies indicate that {S} is at 75-100% surface coverage and of the Langmuir type) or {B} using 0.01 cm3 of the 10-3 to 10-4 M stocks (at this level of addition, the isotherm studies indicate 80-100% surface coverage and Langmuir type). The {C(r)BS} addition series followed a pattern similar to that of the {C(r)SB} series with regards to the agglomeration index, as can be seen clearly in Figure 4a. However, the profile in agglomeration behavior of the {C(r)BS} series differs from that of the {C(r)SB} series as the former shows no clear plateau in agglomeration index over the concentration range studied, even though a higher aggregation index value of nine was observed at monolayer coverage. (b) Outcome of One Component of the LigandReceptor Complex Being Adsorbed ({C(r)S} and {C(r)B}). For one component ligand-receptor adsorption, low indices of aggregation were observed over the ligand-receptor concentration ranges used, and the sample was similar in appearance to the untreated sample in Figure 3b. From the aggregation index profiles for the adsorption of both S and B over the concentration range studied (see Figure 4b), values typically ranged from 2 to 3, with a fall in values only slightly higher than the untreated calcite value of 1.26. The slight elevation in index value may be associated with the surface charge effects that the single ligandreceptor additions have on the agglomeration process.
Figure 4. Aggregation index against ligand concentration for (a) fully labeled, {C(r)SB} and {C(r)BS}; (b) singly labeled {C(r)S and {C(r)B} and rhombohedral calcite.
Habit-Modified Calcite Studies. To explore the role of habit, the following studies on habit-modified calcite were undertaken, using {S} and {B} concentrations set at the optimum aggregation level for rhombohedral calcite, for the {C(r)SB} sequence, ([S] ) 10-6 M and [B] ) 10-4 M in that adsorption order). The overall behavior of rhombohedral calcite and habit-modified calcite is summarized in Table 1, which includes the data for rhombohedral calcite, and general observations are summarized as follows for trends seen in Table 1. It is evident that the aggregation frequency of rhombohedral calcite and hexagonal spindle calcite for the one component adsorption sequence is similar to the respective untreated samples and describes an unaggregated state for these systems. Only the triangular plate habit calcite shows a varied behavior on one component adsorption from the untreated state, which arises since this morphology is assembled in the unlabeled state due to the polar (surface-charged) morphology it expresses. For all three morphologies, the unaggregated nature on single labeling was presumably due to a repulsive interaction arising from the charged nature of the like-covered surfaces. Specific results will now be given. (a) Triangular Plate Calcite. For the triangular plate calcite expressing (001) faces, the following be-
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Figure 5. Micrographs of untreated and when both components of the ligand-receptor complex were adsorbed on to the modified calcite: (a) C(t), (b) C(h)S, (c) {C(t)SB}, and (d) {C(h)SB}. Note that for optical micrographs a-d, the bar is 200 µm. Inset: SEMs of a dried down aggregate, bar 10 µm. Table 1. Observed Aggregation Behavior, Quantified by Percent of Sample Aggregated (Nagg%), Average Number Crystal in an Aggregate (〈Nagg〉), and Average Particle Size of Aggregating Crystals (〈size〉) for All Habits and Labelling Conditions, along with Corresponding Aggregation Indexa sample
Nagg% (σ)
〈Nagg〉 (σ)
aggregation index
〈size〉 (σ) (µm)
C(r) {C(r)S} {C(r)B} {C(r)SB} {C(r)BS} C(h) {C(h)S} {C(h)B} {C(h)SB} C(t) {C(t)S} {C(t)B} {C(t)SB}
43(6)* 42(6)* 53(4)* 78(7) 87(8) 15(2)* 13(1)* 11(1)* 41(5) 65(3)* 20(2) 10(2) 48(6)*
3(1)* 3(1)* 4(1)* 7(1.5) 10(1.3) 3(1.5)* 4(1)* 4(1)* 9(2) 11(1.5)* 3(1) 2(1) 12(1.5)*
1.29(0.06) 1.26(0.06) 2.12(0.04) 6.09(0.10) 8.70(0.10) 0.40(0.03) 0.52(0.01) 0.44(0.01) 3.69(0.10) 7.15(0.04) 0.60(0.02) 0.20(0.02) 5.76(0.06)
29(10) 33(11) 35(12) 35(12) 36(9) 25(8) 17(3.5) 18(6) 17(7) 68(15) 63(16) 65(18) 64(20)
a The standard deviation (σ) of these parameters is given in brackets, and the asterisk indicates that the aggregation profile was similar to the untreated crystals for that morphology.
havior was observed. The triangular tabular-modified calcite on one component adsorption (either {C(t)S} or {C(t)B}) resulted in the crystals disassembling from the column aggregates of the untreated C(t) crystals (see Figure 5a for typical micrographs of untreated C(t)), which are stacked on (001) faces due to polar morphology. This behavior on single component adsorption is reflected in the reduction of the aggregation index (see Table 1), from 7 to below 1. On the adsorption of both components of the receptor ligand, {C(t)SB}, the crystals disassembled from the stacked conformation observed for the untreated crystals C(t), as shown in Figure 5a (i.e., electrostatic-induced aggregation along the [001]
axis) and assembled in an edge-edge configuration shown in Figure 5b, the aggregation index (i.e., the aggregation is directed along the [100] axis using the three (104) faces). With both components of the receptor-ligand complex adsorbed, the aggregation index was found to be 5.7. (b) Hexagonal Spindle Calcite. For hexagonal spindle-shaped calcite with large {110} faces, the aggregation frequency of one component-adsorbed samples, {C(h)S} or {C(h)B}, remains similar to that of the unlabeled samples, C(h), for that morphology, typically less than 0.5 (see Table 1). This reflects a disassembled state for both untreated and one component-adsorbed crystals, see Figure 5c, which shows typical micrographs of a single component-adsorbed sample, with crystals clearly disassembled. Once both components of the ligand complex have been absorbed, the crystal prefers to align along the long [001] physical axes using the carboxyl specific modified {110} faces (Figure 5d). For spindle morphologies, only when both components of the ligand receptor are absorbed, i.e., {C(h)SB}, does the aggregation behavior increase significantly, to a value greater than 3.6. This behavior to ligand-receptor adsorption is similar to that previously seen in the dilution series studies on rhombohedral calcite (see Table 1). Discussion Rhombehedral Calcite Studies. Results from this part of the work suggest that the surface-adsorbed ligand-receptor complexes induced agglomeration, as a result of a nonreversible process, which is dependent on the coverage, the mode of addition, and the mode of binding of the incoming ligand to complete the ligand-
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Figure 6. Mode of binding involving (a) -{BSB}- bridge contact involving two sites on the streptavidin and (b) -{SB}bridge contact involving one site on streptavidin.
receptor formation. For the work on rhombohedral calcite, both ligand-receptor components would be expected to bind to calcite irrespective of any surface stereochemical match between the sorbate and the (104) surface. As previous isotherm studies32 indicate, streptavidin has a coefficient of adsorption K ≈ 106 for calcite, and D-biotin has a coefficient of adsorption of K ≈ 104. Because of their anionic nature of the ligand-receptor components, the low negative surface charge of 0 to -4 mV that calcite possesses in the working 6-8 pH range33 used and the minimal effect single component adsorption has on agglomeration over concentration range used suggest that the influencing mechanism on aggregation of ligand-receptor adsorption is due to an attractive interaction mediated by the surface-supported ligand complex formation. The affinity of conjugate for complex formation and binding to the surface of the calcite was reflected in associated studies, which highlighted the lack of influence of electrolyte addition, hydrogen bonding, adduct inhibitor addition, and temperature elevation on the binding process during the course of this work. (a) Possible Modes of Association Mediated by the Adsorbed Ligand-Receptor Complex. The differing agglomeration index profiles for rhombohedral calcite arising from changing the addition order may be considered in relation to the previous work on adsorption isotherms and relative volume of the two components. Depending on the routes to ligand-receptor adsorption sequence and ligand-receptor complex component concentrations, two geometry situations for ligand-receptor complex supporting agglomeration can be identified. For samples with streptavidin initially adsorbed and subsequently adsorbed with D-biotin, an -{SB}- bridge is envisaged between crystal contacts. This is shown schematically in Figure 6a. This mode of binding would be dependent on the relative difference in volume of the two components and suggests that an incoming biotin would find a site between strepavidins to bind to the incoming calcite surface. This -{SB}bridge arrangement is also envisaged for samples initially adsorbed with lower concentrations of biotin as the level of biotin tagging would generate surface density of biotin, which would leave sufficient available sites for any incoming streptavidin for binding. At higher biotin concentrations where monolayer coverage is approached or achieved, an incoming streptavidin with higher volume would be presented with restricted access to any vacant site on calcite surface. Consequently, with high density of biotin on a pair of crystal contacts, it is envisaged that a second binding site on streptavidin would be used to form a -{BSB}- bridge between crystal contacts. This process of -{BSB}bridge formation is shown schematically in Figure 6b.
Figure 7. Relationship between local ordering and crystal habit. (a) Triangular habit, lattice axis orientation; (b) triangular habit, c-axis orientation of untreated crystals due to polar morphology; (c) triangular habit, a-axis orientation due to ligand-receptor adsorption; (d) hexagonal spindle habit, axis orientation; (e) hexagonal spindle habit, c-axis orientation untreated; and (f) hexagonal spindle habit, c-axis orientation due to ligand-receptor adsorption.
Agglomerate Topology as Crystal Habit Was Varied. Agglomeration geometry was monitored using the variation of the population of face contacts of dried down settled aggregates taken from suspension during tagging. The outcome of this type of analysis carried to investigate the role of morphology on topology of aggregates is now discussed in more detail. SEM micrographs (at 10 µm resolution) were used to compare the short-range order of the aggregates formed by the ligand-receptor adsorption and untreated habit-modified calcites, since differences in contact populations between faces in the dried down state would allude to specific face binding by the ligands. (a) Triangular Plate Calcite. For triangular platemodified calcite, the principal axes in relation to the habit are given in Figure 7a. When untreated, the favored mode of contact between crystals was through the (001) to (001) faces (i.e., alignment along the [001] axis), and when both components are sequentially adsorbed, {C(t)SB}, the favored mode was through the (104) to (104) faces (i.e., generating assemblies aligned along the three [100] of the crystals, leading to sheets), see Figures 7b,c, respectively. It was found that the population of contacts was 95:5% in favor of all (001) contacts for unlabeled samples and 5:95% in favor of all (104) contacts for fully labeled samples. Presumably,
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this dramatic switch of face contacts is a consequence of the polar nature of the habit, along the [001] axis, with a pair of faces exposing either carbonate or calcium ions; thus, oppositely charged in the unlabeled state, this results in an attractive interaction between polar face pairs. Consequentially, this suggests that on ligandreceptor adsorption a preferential adsorption takes place to the calcium rich (001) surface over the corresponding carboxy rich (001) surface by the ligands. This leaves both {001} faces negatively charged, and consequently, a repulsive interaction would exist between the {001} faces in the labeled state. Because of the dominance in the morphology of these faces, this would lead to significant asymmetry in the assembly of the particles. As repulsive interaction would dominate between pairs of negatively charged {001} faces and any attractive interaction due to surface-mediated ligand-receptor formation would dominate along the (104) face, and with both interactions perpendicular to each other, this would lead to a sheet assembly of the crystal. (b) Hexagonal Spindle Calcite. For hexagonal spindle calcite, the principal axes in relation to the habit are given in Figure 7d. The role of crystal habit and selective binding of certain faces by the components of the ligand receptors was particularly evident in the malonate-modified calcite on full labeling, {C(h)SB}, as the population of contacts between (104) and (110) faces increases on labeling over the (104) to (104) face contacts. It was found that the percentage ratios of [(104)/(110)] to [(104)/(104)] contacts for unlabeled samples showed a frequency ratio of 50:28%, whereas with labeling the samples showed a frequency of 77:10%. Both modes of contact gave assemblies of crystals aligned along the [001] axis generating chains of differing topology. See Figure 7e for the proposed mode of assembly for untreated and Figure 7f for the mode of assembly when both components of the ligand receptor were adsorbed onto the hexagonal spindle crystals. However, with both components of ligand receptor adsorbed, the domains of chain were significantly increased. This preference for (104) to (110) contacts on ligand-receptor adsorption of the malonate-modified calcite is attributed to the (110) carboxyl specific facebinding biotin preferentially and the wider atom separations on the (104) allowing preferential binding to streptavidin.
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the underlying crystallographic factors effecting the aggregation of micrometer-sized calcite using conjugated ligand-receptors. Acknowledgment. This work would not have been possible without the support of EPSRC and Unilever Research, PortSunlight, Wirral, U.K., and in particular the contributions from Drs. Andy Waller, Jeff Viro, and Jonthan Warr. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26)
Conclusion From this work, it appears that surface adsorption of streptavidin and D-biotin, was capable of inducing the aggregation of calcite particles of micrometer dimensions, with the habit of the crystals being reflected in the local topology of the aggregates. A future goal would be to use this approach to generate crystal networks and would consequently parallel attempts at assembling particles at the nanometer scale. Also, a considerable amount of future work must be undertaken to resolve the kinetics of the aggregation mechanism based upon the factors controlling aggregation in this study. No attempt was made to model the aggregation kinetics for these systems as the focus of the work was to elucidate
(27) (28) (29) (30) (31) (32) (33)
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