x-ray photoelectron spectroscopic study of the possible interactions of biocells with the surfaces...

SURFACE AND INTERFACE ANALYSIS, VOL. 24, 99-112 (1996)

X-ray Photoelectron Spectroscopic Study of the Possible Interactions of Biocells-with the Surfaces of Select Silicates

Tery L. Barr,’ Sudipta Seal,’* S. Krezoski’ and D. H. Petering’

University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA Department of Materials, and Laboratory for Surface Studies: Department of Chemistry,

Silicate minerals are the dominant materials in the Earth’s crust, and thus literally define the term ‘geochemistry’, but, with the exception of glass chemistry and some areas of catalysis, silicates have, in the past, received scant attention in many areas of chemistry, physics and materials science. In fact, many chemists are largely uninformed about the broad diversity of silicate structures and bondings. Recent explorations by biochemists in the pathogenesis of certain diseases have, however, resulted in a new-found, much broader interest in silicates. This paper extends our studies of the chemistry of mineral silicates using primarily x-ray photoelectron spectroscopy (XPS or ESCA). Following a short consideration of the sometimes contradictory arguments concerning the possible patho- genicity of these materials, we will outline our rapidly expanding unique studies of the interaction of certain biocellular bodies with select silicate systems. The latter studies feature the first ‘before, during and after’ analyses of the silicates (as well as the cells), with the implementation of unique methods for cell-silicate separation and also the freeze drying and surface analysis of the combined systems. In this manner we have been able to identify cell-induced modifications in the surface chemistry of the silicate components, along with alterations in the cellular species. Many of these analyses were facilitated by the recent clarifications in bioorganic ESCA.

INTRODUCTION

It is reasonably well established that many of the most virulent diseases experienced by humans occur due to overt exposure to a variety of silicate minerals, particularly during respiratory processes.’ This has led to the alteration of practices, enactment of programs of removal and replacement, and the virtual creation of major US government agencies, e.g. the EPA. In many instances fear and misunderstanding have combined with a lack of knowledge to create situations for silicate minerals that border on ‘witch hunts’, with both exposures and castigations of the culprits. In some circles it has been assumed that essentially any material containing Si is a poison. All of this is promulgated on a planet whose crust and mantel are, and, as far as man’s exis- tence is concerned, always have been more than 90% silicates. How man survived (and positively evolved) during several million years of living in the caves, gullies, savannas and deserts of Africa is inexplicable if everything from crushed sand to common clays is extremely pathogenic. Despite these obvious excesses, however, it cannot be denied that some silicates (e.g. the amphibole crocidolite and the sheet serpentine, chrysotile) are harbingers and initiators of very virulent, and in some cases deadly, diseases.’ All of this suggests that it is time not only to study and understand the diseases themselves and their pathogenesis, but also the silicates themselves and particularly their surface chemistry. Common-sense dictates that if silicates are

* E-mail address: [email protected]

CCC 0 142-242 1/96/020099- 14 0 1996 by John Wiley & Sons, Ltd

involved then their surfaces will substantially influence the origins and growth of these maladies.

For these reasons we have recently begun the first concerted, detailed study of the bulk and surface chemistry of key silicates, before, during and after interaction with various bio-cellular bodies. These studies have included examples where the silicate-cell interaction has been ‘cultured’ under various conditions and for varying periods of time, followed by the separation of the components3 and finally the ‘cleaned off silicates have been characterized (by various forms of microscopy, XPS and MAS-NMR). Also we have examined cases where the pretreated cells have been freeze dried and similarly analyzed. In addition, several mixed cell- silicate systems have been centrifuged, and the combined material freeze dried and characterized by XPS. Also the cells separated from the silicates were freeze dried and analyzed by XPS.3

In the present paper, we present a summary analysis of the results achieved so far for silicates, cells and mixed systems, with an emphasis on the XPS-revealed surface chemistry. Several of the key systems will be described in some detail to try to convey to the reader a perspective for the methodology being employed.

EXPERIMENTAL

Materials

Samples of silicates were obtained from a variety of representative commercial and natural sources. For example, samples of serpentine asbestos (e.g. chrysotile)

Received 7 June 1995 Accepted I November I995

100 T. L. BARR ET AL.

and amphiboles (e.g., cummingtonite) were obtained from typical geological sources and provided by Dr G. Mursky of the Earth Sciences Department of the Uni- versity of Wisconsin-Milwaukee. Some of the samples obtained are fibrous, as has been demonstrated by optical microscopy. Other samples exhibited plate-like or flaky textures. Based upon our analyses and observ- ations, we determined that these samples have under- gone the expected modest environmentally induced alterations of their surface character, e.g. surface- oriented non-crystallinity, adventitious carbon deposi- tion, outer surface aquation, etc. It should be noticed that these problems were found not to produce variations in configuration and composition outside of the uncertainty range needed to bracket the general range of suppositions reached in the present study. In our preliminary surface analysis by XPS (or ESCA) of cell- silicate interactions, small quantities of chrysotile and cummingtonite fibers were pressed as wafers under 6500 psi. These wafers were configured as thin 6 mm x 4 mm rectangles for ready insertion into the ESCA instrument (see below).

Techniques used for the cell-silicate interaction study4

Sterilization. A typical flow chart of sterilization is shown in Fig. 1. The bottle and the sample (silicate) were sterilized by autoclaving at 250 O F for 15 min.

Cells. Ehrilch cells, a mouse (murine) tumor line, were used for cell culture. Cells were grown on various sub- stances as a monolayer in a MEM (minimum essential medium) containing 4% fetal bovine serum. The MEM’ consists of a complex mixture of salts, proteins, vitamins

and sugars in a buffered H,O solution. Cells were realized from the monolayer using 1.0 ml of 10 x trypsim- EDTA solution (Sigma) in a tissue culture flask (75 cm’). These cells were pooled in a bottle containing fresh medium (MEM + 4% bovine serum) and divided into several (100 mm diameter) culture plates. Sterilized silicate fibers were added to these plates to cover 20-30% of the surface of the plates. The other plates with cells were used as controls. An 8 ml aliquot of the medium was used in each plate. The following experimental conditions were used: cells alone in medium; cells + silicate + medium; silicate + medium; silicate. All of these experimental protocols were kept in a Forma Scientific Incubator for 6-7 days at 37°C in order to induce cell culture. A mixture of 95% air and 5% carbon dioxide was used as the gas medium. The cell medium on the plates was changed every 2-3 days. The cell growth on the silicates was periodically examined using an inverted microscope. The average cell concentration was found to be 3 x lo4 cells ml-’, with the silicate fiber concentration being 12.5 mg ml-’.

Separation procedure. For the wafer samples, distilled water washing was employed prior to analysis of the cells on the wafer. Several washings were done to remove as much of the cell material as possible while simultaneously retaining a ‘living’ cell growth. The cells were released from the fibers using 10 x trypsim solution and the suspension was transferred from the culture plates into 50 ml centrifuge tubes using MEM. Then the fibers were allowed to settle by gravity for 1-2 min and the cells in suspension were decanted into another 50 mi centrifuge tube. The fibers were again resuspended into choline containing phosphate-buffered saline and allowed to settle. The cells in suspension

Figure 1. A schematic diagram of the sterilization process used in the cell culture experiments

XPS STUDY OF BIOCELL-SILICATE INTERACT10 N 101

were transferred to a fresh tube for separation. The fibers were rinsed with distilled water to remove as much as possible of the remaining cells (see the results below).

Cells from the 'cell alone plates' were similarly released as described above, using the trypsim solution, washed into a 50 ml centrifuge tube and concentrated by centrifugation for 5 min at 2000 rpm. The separated fibers were washed with distilled water and then dried in an oven at 100°C. All of these systems were then subjected to XPS analysis. It should be noted that the cells alone and the cells separated from the fibers were also subjected to XPS.

Freeze-dried samples. In several cases cell-treated silicates were removed from the incubator after 6 days, rinsed first with a phosphate saline buffer (pH 7.4) and subsequently with 0.25 M ammonium bicarbonate to reduce any salt effects, then frozen on dry ice for 5-10 min and finally freeze-dried at a pressure of lo-' Torr in a Lab- conco freeze dryer. Similarly, cells treated with chrysotile and cummingtonite were also freeze-dried under the same conditions. These samples were also subsequently subjected to XPS analysis.

Characterization techniques (XPS or ESCA)

A few of the ESCA results were recorded on a Vacuum Generators (VG) ESCALAB system. A conventional AlKa anode was employed for the source, producing a binding energy scale specified by Au 4f,,, = 83.98 - + 0.05 at a line width of 1.0 eV. The background pressure during analysis was -1 x Torr. Most of the other studies were performed on a Hewlett-Packard (HP) 5950 ESCA instrument using a high-resolution x-ray monochromator. The latter system produces a similar Au 4f7/, peak structure at a slightly higher resolution. All of the materials examined were insula- tors, and thus produced distinctive charging shifts. The latter were removed and the binding energy scale established by using a combination of electron flood guns and the fixing of the C 1s binding energy6 of the hydrocarbon part of any adventitious (hydro) carbon line at 284.6 eV. This well-established procedure has been successfully employed by us in numerous previous studies of silicate^.^^' The H P system was somewhat more amenable to the latter process.

X-ray photoelectron spectroscopy analysis. It should be noted that substantial use is made in the following of the modest, but progressive, shifts in the measured XPS binding energy peak positions. Assuming, as we have mentioned above, that we have provided a reasonable fixture for the zero of the binding energy (Fermi edge), we still must provide some justification for our subsequent use of arguments that, in effect, equate experimental shifts to initial state (chemical) differences. We recognize that the measured positions of the binding energies are significantly influenced by (measurement induced) final-state effects. However, we have determined that to a great extent these final-state features cancel during comparisons of shifts for similar oxide systems, and although the latter is, of course, not an absolute statement, the residual effects are readily

accommodated inside the general arguments of the present study.'-''

RESULTS

X-ray photoelectron spectroscopy investigations of various silicates

We begin the discussions of our silicate XPS results with a short synopis of our ongoing studies of zeolites and related framework silicates." The latter materials are characterized by the formation of three-dimensional TO, tetrahedra, where T is usually Si but may be Al, and where every corner-oxygen is shared with one other tetrahedron (see Fig. 2). When A1 is substituted for the Si, as is the case of feldspar (the most plentiful mineral in the crust of the earth) and also for almost all zeolites, there must also be a cation (e.g. Na') for every alumin- ate (AlO,-) unit. The latter are found selectively lodged in various cavity sites in the framework lattice. Detailed XPS studies of these materials readily demonstrate that the resulting Si and A1 binging energies differ for most of these systems in progressive, reproducible shifts (see Table 1).7-9*12 In this manner, shifts for the framework silicates were found to depend on the [Si]/[Al] ratio (for all cases with A1 in the tetrahedral lattice) and/or on the change of the co~n te r - ca t ion .~* '~ . '~ Thus, the shifts have been related to changes in the degree of covalency/ ionicity of the framework cations and the induced charge of the counter-cation~.~-~ The characterization method is found not to be able to detect fairly subtle

LJu

a-Framework b. Sheet (Clays)

Si206 n

c.Double chain (Amphibole) Figure 2. Rendition of tetrahedral sublattice for silicates. Large open circles represent oxygen; closed circles are tetrahedral (generally silicon) cations. Adapted from Ref. 19.

d. Chain ( Pyroxene)

102 T. L. BARR E T AL.

Table 1. Binding energy shifts for various framework alumino- silicates and silica referenced to C 1s = 284.6 eV"

Material Si/Al (tet) Si 2p Al 2p

Zeolite Na-A Zeolite lo-sod Aluminosilicate sodalite Blue ultramarine Zeolite Na-X Analcite Pink ultramarine Zeolite Na-Y Zeolite ZSM-5 Pure siliceous sodalite Silica

1 .o 1 .o 1.05 1.20 I .25 1.5 1.5 2.5

30 Q)

Q)

101.1 101.4 101.5 101.7 101.95 102.2 102.2 102.55 103.1 103.2 103.5

73.5 73.5 73.6 73.85 73.9 74.0 74.4 74.2 74.7 - -

'The table demonstrates the progressive increase in Si 2p and Al 2p binding energies. tet, tetrahedral.

Table 2. Binding energies and, in parentheses, corresponding linewidth ( f 0.05 eV) for representative sheet silicates"

Materials Si/AI SI 2p Al 2p 0 1 s MgZp

Allophane 1.0 102.3 74.3 531.75 -

Kaolinite 1.0 102.45 74.3 531.5

Montmorillonite 2.0 102.75 74.8 532.0 -

- (2.45) (2.7)

(2.25) (2.2) (2.45)

(2.05) (1.98) (2.36)

Si/Mg

Chrysotile 0.67 102.5 - 532.0 50.0 Antigorite 0.67 102.4 - 531.6 49.95

- 532.2 50.5 Talc 1.33 103.1

Binding energies are referenced to C 1 s = 284.6 eV.

differences in structure, e.g. that between zeolites and feldspathoids of similar [Si]/[Al] ratio,14 but the method readily detects impurities such as octahedral alumina unit^.^^'^ As a typical example of the utility of the XPS method we have noted that the dealumination procedures known to extract A1 out of the tetrahedral framework of a Y zeolite often discard the byproduct as surface-oriented waste and the latter are easily identified by XPS as A1,0,

Unlike the framework silicates, the sheet variety (micas, clays, etc.) share only three silicate corners, forming an infinite two-dimensional lattice and exhibiting an [O]/[Si] ratio of 2.5 (see Fig. 2)." The remaining tetrahedral (apical) oxygen is stretched across a plane perpendicular to this tetrahedral subsheet to form a couple to a companion octahedral subsheet that runs parallel to the tetrahedral subsheet. The octahedral sites are generally occupied by either Mg (tri-octahedral) or A1 (di-octahedral) units that resemble brucite or gibsite, respectively.' This double-layered 'sandwich may be complete or, in some cases, another tetrahedral subsheet may grow on the outer side of the octahedral layer. The first case is referred to as 1 : 1 and the second as 2: 1. Generally, nature does not provide pure SiO,,, tetrahedra layers or pure MgO or A1,0, octahedral subsheets, i.e. numerous cases exist with various amounts of A10,- in the tetrahedral layer and Mg2+, A13+, Fe2+, Fe3+ and other cations in the octahedral layer. l1 When charge balances are needed, counter- cations are once again found trapped in the resulting structure, usually in counter layers in-between the stacks of the previously described 'sandwiches'.' When counter-cations are not present these sheet silicates tend to be soft or slippery, (e.g. talc, kaolinite and most smectites), but when the sheet silicates contain large quantities of counter-cations they become noticeably harder and brittle (e.g. many micas).' '

In the recent past, our XPS studies of sheet silicates have concentrated on the di-octahedral clay min- e ra l~ . ' . ' ~ - '~ We have found the binding energy results to be reproducible (Table 2) but quite different from those for the framework silicates, with similar [Si]/[Al] r a t i o ~ . ~ * ' ~ , l Once again we find progressive upward shifts in Si and A1 values with growth in the [Si]/[Al] ratio, but in this case the effects are definitely muted,

since they are felt primarily across the apical oxygens between relatively contiguous subsheets.' 4*1

As part of the present XPS study we have now expanded to investigations of the tri-octahedral (Mg- containing) sheet silicates (see Table 2 and Fig. 3). Talc, a 2 : 1 tri-octahedral, is revealing in that it continues the previously noted progression where in comparison with chrysotile an increase in the [Si]/[Oct] ratio results in an increase in the binding energy of both the Si and octahedral layer cations. These arguments, however, are not completely followed by the silicates in question, in that talc with an [Si]/[Mg] ratio of 1.33 exhibits a lower Si 2p binding energy than montmorillonite, the di-octahedral 2 : 1 clay with [Si][Al] -2. This question will be addressed further in a more detailed discussion of the tri-octahedral sheet silicates presented elsewhere.'

Chrysotile (white asbestos) is a previously unreported XPS investigation of major importance in the present study because one of its structural forms is the most common asbestos in commercial use." Before we proceed we should address again with some care the generalities of this area of materials science and engin- eering which we find fraught with misinformation and innuendo. First, it should be noted that chrysotile is one of the common forms of the sheet silicates in a class labeled as serpentines." The latter refer to a group of magnesiosilicates of general formula Mg,[Si,O,](OH), . There are three principal species in this group: chrysotile, antigorite (also described herein) and lizardite.' The microstructures of these materials tend to differ from one another in subtle structural ways that end up reflected macroscopically in a more obvious fashion, e.g. chrysotile is often found in nature as silky

0 4

m 6 -

1 inner h y droxyl

Figure 3. Representative depiction of section of typical sheet silicate.

XPS STUDY OF BIOCELLSILICATE INTERACTION 103

fibrous veins, while the macrostructures of the other two are usually similar to layered kaolinite. It is, of course, the silky (asbestiform) fiber nature of the chrysotile that makes it an excellent choice for such commercial uses as brake linings and building insulation.” White asbestos is found to have a higher heat resistance than ‘blue asbestos’ (its amphibole counterpart; see below) but the latter has a greater tensile strength resulting in some selectivity in the type of asbestoses employed for particular jobs. All of this has now been totally reversed, however, due to the discovery of the serious health hazards resulting from exposure (particularly, but not exclusively, from inhalation) to these asbe~toses .~ ,~ ’ The problem with this observation from a materials point of view is that in trying to associ- ate ‘cause’ to this ‘effect’ investigators in the biomedical community have identified three probable sources : the fibrous nature of the asbestoses; the presence of various iron components; and a combination of the two. This has led to a number of overt statements and misrepre- sentations. For example, numerous biomedical studies refer to chrysotile as an Fe-containing mineral,, when in fact geochemists have confirmed, over and over that chrysotile contains essentially no iron.” The 1 : 1 minerals that resemble chrysotile, and contain iron, are primarily berthierine or cronstedtite, and although similarly structured, these iron-containing materials are not true serpentines.” The aforementioned lizardite (L) and antigorite (A) are indeed serpentines and, therefore, have been castigated along with chrysotile in the environmental science literature, despite the fact that neither L nor A are generally fibrous asbestiforms, nor do they contain Fe. In addition to these discrepancies it is largely ignored that the overt use of the ill-defined term ‘asbestos’ has been attached to a wide variety of materials, some of which are apparently not even pathogenic. Before we leave this issue we should note that although chrysotile itself does not contain Fe, it is generally produced in nature from olivine(s) [Mg,(Fe,)SiO,], which are common mantle rock structures that do feature lots of Fe. As a result, chrysotile deposits may be accompanied by magnetite (Fe,O,).” It may be the presence of the latter that has been detected by bioscientists.’

We have employed XPS to analyze, in detail, both chrysotile and antigorite (Table 2 and Figs 3 and 4). One of the important results is the near-reproducible match of the binding energies and line widths of the resulting peaks for both materials.17 This is a further reflection of the ability of the ESCA system to track (in detail) common chemistries, and correspondingly the general lack of capability of ESCA to discern modest differences in ~ t r u c t u r e . ~ ” ~ The principal uncertainty in these results is the substantial increase detected in the surface (compared to bulk) [Si][Al] ratio for both serpentines. The explanation for this, for chrysotile, may rest on its spiral nature” that may selectively bury the octahedral sublayer in favor of the tetrahedral units, but this explanation does not hold for antigorite. On the other hand, the fact that the linewidths of both Si 2p and Mg 2p are uniformly narrow for both these systems seems to preclude any appearances of extensive surface byproducts, thus belying any contention of surface alteration. This feature is still being examined.

As a continued form of this type of analysis we have

536 534 532 530 528

Binding Energy (ev)

L

106 104 102 100 98 56 54 52 50 48

Binding Energy f e w Binding Energy (ev)

Figure 4. Key XPS peak structures for representative chrysotile.

employed XPS to investigate a number of the double- chained (amphibole) silicates with general formula A,- ,B,M,T,O,(OH), . 1 1 * 1 8 In this case the designation ‘double chain’ refers to the arrangement of the tetrahedrally bonded (silicates or T-containing) unit, i.e. as in Figs 2 and 5, where one should note the double set of T, and T, sites. These tetrahedral units are thus only 4 T across in the page, but stretch to near-infinite chains in and out of the page. The T sites are typically filled by Si, but may contain other species, particularly Al. Just as in the case of the framework silicates, when the latter are present there are sometimes counter-cations (e.g. Na+) in the A sites. The M sites are corresponding octahedral sites that can be seen to ‘cement’ two double- chained tetrahedra together into a complex ‘sandwich’. This sandwich is very resilient inside itself. Thus the darkened regions of Fig. 5 are deemed so tough by geochemists that they are commonly referred to as the ‘double anvils’.Ig Thus, whereas, elemental exchange is relatively common inside the double anvil, breakage and ‘sectional extraction’ generally only occur on a zigzag course around these units.”~” The M,, M, and M, sides tend to be occupied by typical octahedral cations similar to those found for sheet silicates , e.g. Al, Mg, Fe2+ and Fe3+.” The M, site is the fundamental coupling site tying the octahedra to the tetrahedra. It is occupied by typical counter-cations ranging from Mg2 +

(the most covalent) to Na+ (among the most ionic).11g18 A number of typical amphibole examples have been

provided by our geochemical sources and examined with XPS. These are identified in Table 3 along with the approximate surface compositional formula as determined by the relative quantitative analyses realized by


Figure 5. Structure of amphiboles. The tetrahedral double chains yield two distinct sites, T, and T,. There are three crystaltographically distinct octahedral metal sites: M, is cis-bonded to two OH, M, is trans-bonded to two OH; M, is not bonded to OH. The large 6- or 8-coordinated M, metals flanking the ribbon cross-link adjacent sandwiches.”

XPS. Also found, but omitted in the latter formulations, are small concentrations of lesser elements.,

Also presented in Table 3 are the average binding energies realized in this study.3 It should be noted that, as in the case of the framework and sheet silicates, there are no single values for these amphibole systems, but there are (as described below) repetitive, but explicable, shifting chemical patterns exhibited by each ‘type’ of amphibole. One feature, however, that should be apparent is that these shifting patterns realized by the amphiboles are far too complex to be understood in terms of the simple M-0-M’ covalency/ionicity arguments employed for the framework silicates. But ramifications of that principal argument still seem to apply to these double-chained silicate forms. In our view, the key to the present analysis lies in the relative covalency/ ionicity of the species that occupies the M, position (see Fig. 5 ) and how this element couples through oxygen to the lattice cations in the MI , M,, M,, T I and T, sites.,

Thus, in the cases of the tremolite and anthophillite systems, the only significant difference between the two is the change from Ca in M, for the former to Mg in M, for the latter. The Ca-0 bond is more ionic than the Mg-0 bond (some readers may wish to substitute cation size here for bond strength) and this enhanced

ionicity is translated (on the other side of the oxygen) into enhanced covalency (significant reduction in binding energy) for both the tetrahedral Si-0 bonds and the octahedral Mg-0 bonds.,

The results for the other amphiboles in Table 3 are more difficult to interpret, particularly in view of the obvious complexity of the composition of the subunits of the resulting ‘double anvils’., It should be noted in Table 3 that all of the other amphiboles exhibit surface composites with substantial quantities in site M, of the more ionic counter-cations, i.e. CaZ + and Na’ (as opposed to Mg”). This fact should contribute substantially to the general downfield shift in the binding energies of the cations in these systems compared to those in anthophillite., These binding energies seem to be particularly low for the actinolite materials. This may be a result of the significant presence in the latter of K’, a species which when doubled in amount substantially exceeds the ‘field’ influence of Ca”. In the case of glaucophane, we also find a particularly large relative amount of Na’. This feature seems to contribute to the most substantial downfield (enhanced covalency) shift yet observed for the lattice, Si, Al, Mg and Fe species in these amphiboles, see Table 4.9

It should be noted that the shifts detected are also in part due to the changes in relative distribution of lattice

Table 3. Chemical formulae and elemental binding energies of the amphiboles as obtained from ESCA results

Mineral Formula

- 103.45 50.95 50.5

- - Anthophyllite Mg2[Mg61[Si,0,,l(OH)2

Tremolite Ca2[M g6l [ s i 8 0 Z Z l ( H, 2 - 347.5 49.7 - 102.7

Hornblende (Car M ~ ) [ M ~ ~ , 2 F e ~ ~ A ’ l . ~ I [ S i 6 A ~ Z o 2 2 1 [ o H , c112 - 347.9 50.8 711.9 102.6

Cummingtonite (Na, Ca, Fe,.,)[Mg,.,Fe,,AI, ,][Si6,,AI,.8022](OH) - - 50.25 710.7 102.2

Actinolite (K Ca)2CMg4.,Fe,.,lCSi,.,Al, ,,02,1COHr FI2 293.3 347.55 50.1 710.9 102.6

A I Z D 0 1s

- 532.6

__ -

73.5 -

73.5 -

74.5

74.2 73.85 531.6

XPS STUDY OF BIOCELL-SILICATE INTERACTION 105

cations within each substructure (e.g. changes in the [Si]/[Al] ratio inside the tetrahedral sublattice). Thus the general reduction in the amount of octahedral A1 and reduction in total Fe may contribute to making the Mg-0 bonds in actinolite-X more covalent, i.e. reducing the Mg binding energies.

Perhaps the most important feature detected in these amphibole XPS studies is the certain appearance of two different types of A1 species that have been reproducibly found for several of the systems (see Fig. 6).3 The most difficult part of this stage of the analysis is the identification of the origins of those two species. Based upon comparisons with the previously reported A1 results for the framework and sheet silicate^,^^'^^'^-'^ and the bulk values reported in Deer et d.," we have tentatively made the octahedral us. tetrahedral assignments listed in Table 4. The relative distribution of these two A1 species will play an added role in the studies reported

74.0

I

(a) Y -Jmm

a6 ar a2 ao 78 76 74 72 70 6a

Binding energy (eV)

Figure 6. The Al 2p spectra for select amphiboles and alumina. Note the double peaked structure of the former.

Table 4. Assignments of the aluminum species detected in the amphiboles examined by XPS

Al in octahedral Al in tetrahedral sublattice sublattice

Hornblende 74.5 73.4 Cummingtonite 74.4 73.4 Actinolite-X - 73.85 Actinolite-hornblende 74.4 (weak) 73.8 Glaucophane 74.1 5 -

below for the interaction of cummingtonite with mouse tumor cells.

Along with the core-level binding energies and line widths described above, it should be noted that we have found that XPS-induced valence band and loss spectra played important supportive roles in the interpreta-

In addition to the aforementioned studies, we have initiated XPS investigation of the ortho and single-chain silicates. These results will be described elsewhere.

tions.8% 10.20,2 1

X-ray photoelectron spectroscopy investigations of select silicatecell interactions

General statement of practice. As previously described, a principal thrust of our studies has been to try to deter- mine key features in the interactions of living cells with the surfaces of various silicates. The program has, so far, been restricted to the implantation of murine (mouse) tumor cells onto two types of silicates: the sheet- serpentine system, ~hrysot i le ; '~ and the amphibole cum- ming t~n i t e .~ The reasons for these choices are that both materials exhibit obvious propensities for fiber formation, and the XPS study of our untreated cummingtonite revealed a substantial presence of Fe species despite contentions in previous bulk analyses that cummingtonites are not prone to substantial amounts of iron.

Silicates isolated following various treatment protocols. AS outline above, both cummingtonite and chrysotile were treated in a particular protocol with mouse (murine) tumor cells. Such a procedure also involved a variety of treatment steps and the necessary inclusion of a MEM as the growth 'vehicle' for the cells.4 For this reason it was necessary to examine the silicate with only the MEM present and also following each of the procedural steps without both cells and MEM. The list of the results are presented in Table 5 for both cummingtonite and chrysotile. It should be noted for both 'parent' materials that the resulting binding energies vary a bit, but even more dramatic than these slight alterations is the general overriding similarity of all of the numbers. Thus, it seems reasonable to state that subjecting both cummingtonite and chrysotile to the various forms of autoclaving, incubation, washing, drying and even doing this while immersing these silicates in the MEM produces little, if any, change in the silicate chemistry, as indicated in the resulting binding energies. In a similar way we have tried to follow the relative elemental concentrations for the two silicates during these same exposures. The results of these studies are presented as parts of Tables 6(a) and 6(b). In this case the values are not determined in an absolute sense, but rather relative to an arbitrary fixed value for Si. In these cases certain small alterations are noted, particularly during the treatment of cummingtonite, but these results can, we feel, be largely attributed to the significant uncertainties of this type of measurement. In the case of the chrysotile" there may be a slight rise in the very modest Fe concentration detected with addition of the medium, but this change must also be viewed with some caution, particularly because of our previous contention that most, if not all, of the Fe in any chrysotile


Table 5(a). Binding energies (in eV) for cummingtonite subjected to various treatmentsa

With cells Separated With Fresh A. W. D A. I. W. D (freeze-dried) from cells medium

Si 2p 102.2 101.5 102.1 (2) No Si-0-C

Mg 2p 50.0 -

Fe 3p 55.4 -

55.5 (2)

0 1 s 531.4 531.2

Fe 2p 709.7 (2) 71 0.8 Al 2p 74.45 73.6

74.4 (2) 73.4 73.65 (2)

102.3 101.4 102.1 ? 102.2 103.2

(Si-0-C) - 49.6 ?

531.3 531.3 531.5 531.4 55.8 55.35 54.8

-

71 0.8 71 1.2 71 0.0 74.2 74.7 73.9 -75.0 73.4 73.? 73.8

"Abbreviations: A, autoclave; W, wash; D, dry; I, incubation; (2). second run

Table 5(b). Binding energies (in eV) for chrysotile samples under various stages of treatment"

With cells, With cells, With cells, then cleaned then cleaned then cleaned Freeze dried

Raw With medium ( 1 ) ( 2 ) (3) with cells

Si 2p 0 1s Mg 2P Al 2p Ca 2p Na 1s Organic units Fe

102.6/102.5 102.4 102.8 532.0 531.8 532.1 5 50.0 49.75 50.25 73.5 small 73.4 moderate

347.5 small 347.05 347.5 small

None N-C=O N-C - 1071.2 1071.55 weak

71 0.65

a 1 , 1 st run; 2, 2nd run; 3, 3rd run

102.4 102.4 102.55/103.7 531.85 531.75 531.8 49.8 49.6 50.1 - No None - -

1071.25 1071.3

71 1.3 N-C=O -O(?) N-C=O

system may arise not from the chrysotile itself but rather from some iron-containing byproducts of the chrysotile production."

Dramatic changes in the XPS results seem to occur following cell growth on these silicates (and subsequent cell Both the binding energies and relative elemental distributions experience these changes. First in Table 5(a) we note that the A1 2p for the cummingtonite, which readily revealed two peaks before and during the 'preliminary' treatment, now after removal of the presence of the cells exhibits largely one recognizable peak. The binding energy of the latter A1 peak suggests

predominantly that the A1 is either located in the tetrahedral subsheet or results from A1,0, formed during the interaction. Thus, there seems to be a binding energy shift for both of the lattice A1 species, perhaps reflecting the changes in elemental distribution revealed in Table 6(a). Also in Table 5 there seems to be finite changes in the binding energies of the resulting Mg and Fe species. In view of the possible movement of some of the lattice A1 to the surface as A1,0 , , it is not a surprising feature for the other octahedral cations also to shift in binding energy to compensate for the removal of Al. In addition to the changes in binding energies, Table

Table 6(a). Relative quantification of cummingtonite before and following various treatments designed to study the cell-silicate interaction: results generated from 0-200 eV ESCA survey scans with respect to Si 2p (30 units)"

Sample Al 29 Fe3p Mg 2p

Cummingtonite (plain) : VG ESCA Cummingtonite (plain): HP ESCA Cummingtonite (A, W, D) : HP ESCA Cummingtonite (A, I, W, D ) : HP ESCA Cummingtonite (A, I, M, W, D ) : HP ESCA Cummingtonite (A, I, M, C, W, D ) : HP ESCA Cummingtonite (A, I, M, C. W, D ) : VG ESCA Hornblende (plain): VG ESCA

10 12 4 9 10 4.2

14 4 7 1 1 4 9.6 12.8 4

21.5 14 6 16 15.5 5 11 8.4 5.2

-

'Abbreviations: A, autoclave; I, incubator; M, medium: C, cells: W, wash; D, dry.

107 XPS STUDY OF BIOCELL-SILICATE INTERACTION

Table 6(b). Relative ESCA quantification of chrysotile before and after various treatments designed to study the cell-silicate interaction: results generated from 0-200 eV VG ESCA survey scan with respect to Si 2p (30 units)a

Materials Fe 3p M g 2~ SI M g b

Chrysotile (plain) 2.2 9 1.2 Chrysotile (A, M, I, W, D) % = 8.75 1.3 Chrysotile (A, M, C, I, W, D) 0 % ' 7.5 1.5

"Abbreviations: A, autoclave; I, incubator; M, medium; C, cells; W, wash; D, dry.

Ideal values of Si/Mg = 0.67. % , increase relative to 2.2.

6(a) reveals that the various lattice cations in the presence of the cellular species experience quite significant changes in relative distribution. There seems to be created, for example, a distinct rise in the surface visibil- ity of Al, Fe and Mg relative to the detected Si, with the rise in A1 particularly dramatic, suggesting that most of it is now on the surface of the ~ i l i ca t e .~

In addition to these changes in the binding energies of Al, Mg and Fe, there is an interesting lack of change in the Si 2p spectrum for cummingtonite after interaction with the murine cells and subsequent removal of the organic material^.^ When the cummingtonite-cell combined (freeze-dried) system is examined by XPS, as expected, all of the silicate peaks are muted in size due to the surface presence of the cellular material.3 In addition, the freeze-dried systems exhibits the apparent retention of the double A1 peaks in approximately the same ratio as detected previously for the higher binding energy to lower binding energy forms. The binding energies for these two peaks are, however, both - 0.4 eV lower than the corresponding peaks detected previously for the cummingtonite system. The major differences experienced by the lattice cations for the cell-bound cummingtonite occur in the complexity of the Si spectra (see Fig. 7). In this case several different samples repeat- ed a similar three-peaked manifold. The linewidth of this manifold for cummingtonite (2.07 eV) is indicative of the presence of several peaks grouped together. (Note that the corresponding 0 1s manifold has a linewidth of

- 2.0 eV, while for all other cummingtonite systems the linewidth for 0 1s is, at least, 20% broader than that for Si 2p.) The largest part in the Si 2p manifold of peaks occurs at -102.2 eV, i.e. in the same area as 'unper- turbed' cummingtonite. The other two peaks are at - 103.2 and - 101.4 eV, respectively. In view of the fact that this reproducible structure seems to be reserved for mixed cases of silicates and cells, we propose an explanation (presented below) that is based on a chemical interaction involving the creation of Si-0-C bond^.^.'^

The results for the freeze-dried system of chrysotile murine tumor cells have a striking resemblance to those for the cummingtonite-cell cases. In the chrysotile case, the oxygen and magnesium spectra largely duplicate the features for the pretreated chrysotile. At the same time there is a manifold of Si 2p protrusions with the principal peak at 102.45 eV in the vicinity of that realized by the Si for untreated chrysotile. However, just as with the cummingtonite--cell case, there is for cell-coupled chrysotile a broadening of the total Si 2p manifold with the ready appearance of at least a second peak at - 103.8 eV. This binding energy position is identical to that detected when graphitic carbon is interacted with 50, to form well-demonstrated Si-0-C type bonds. The distribution in this case suggests that the degree of formation of this direct chemical interaction between the silicate and cells is less pronounced in the case of chrysotile than it is for c~mmingtoni te . '~

Examination of the cells by bioorganic ESCA Pre-cell adventitious carbon. In all of the systems under XPS investigation we were able to detect a variety of carbonaceous species. In the case of the silicates that had not been exposed to any organic units, the carbons realized were the species that are generally designated as adventitious carbon. This ubiquitous material played an active role in the present study (and in many related investigations) through its use in establishing the binding energy scale. In previous publications several of the authors have described in detail adventitious carbon and its use as a binding energy standard.6s8 It was therein noted that the principal component of this mixed carbon system is almost always a series of airborne hydrocarbons that tend to absorb onto all air-exposed surfaces in a

Si (2p) spectrum

Unaltered cummingtonite

I 1

116 112 108 104 100 96

Binding energy (eV)

Figure 7. The XPS Si 2p spectrum for cummingtonite freeze-dried with biocells after extensive interaction.


manner such that these hydrocarbons are usually outer surface specific and well dispersed throughout the host surface. When properly ‘oriented’ these materials have been shown by numerous investigators to have a relatively uniform binding energy of slightly less than 285.0 eV.” For purposes of uniformity we generally select the value of 284.6 eV for ‘typical’ adventitious carbon. It should be noted, however, that several groups (including our own) have observed that not all C,H, and carbon-only systems should be assumed to have a singular binding energy. Some evidence exists for a progressive growth in C 1s with complexity of aliphatic hydrocarbon systems (up to -285.3 eV) and a decrease in C 1s with complexity of any aromacity (down to - 284.4 eV for gra~hi te) . ’~ With these complications in mind one may still use ‘average’ values such as 284.6 eV for C 1s for our representative C,H,, and in the present case we have done so for the hydrocarbon part of any adventitious carbon and any hydrocarbon features of our cellular species. When one does this, it is readily apparent that adventitious carbon is rarely all hydrocarbon. Due to the presence of CO,, CO, alcohols and various carbonyl-containing organics in our environ- ment, one generally finds C 1s spectra similar to those in Fig. 8(a, b) for most air-exposed materials. It should be apparent that the shoulders on the high-binding- energy side of these spectra are due to the ‘residues’ of these various ‘oxidized carbonaceous species. For a long while little could be said with certainty to identify these species, but, primarily due to the recent appearance of the book entitled High Resolution X P S of Organic Polymers by Beamson and most of this uncertainty has disappeared. The Beamson and Briggs (B&B) study was performed with great care using a Scienta ESCA 300 and with superb control of the binding energy scale. As a result, the extensive binding energy tables that appear as appendices in the

I . . ’ . ’ . . .. . . . . .. . . . .

I.

(c) Cell + C-mte freeze dned

I

I ‘ t I , I 1 I ,

I

291 289 2 i7 2 k 283 281 t

Binding Energy (eV)

Figure 8. The XPS C Is spectra for select amphiboles. Note that only (c) exhibits extensive C-N type peaks.

B&B bookz4 provide excellent registers of the peak positions and ranges for almost all of the common carbonaceous bond situations that are realizable. Using these tables, and our own data, we have therefore been able to evolve a relatively detailed analysis for adventitious carbon (AC). This is absolutely necessary because this ubiquitous material is always present, and one can only realize a fairly detailed description of the carbonaceous surface species produced in our interactions of silicates with cells if one is able to continuously identify and, in effect, separate out the contribution of AC.

Fortunately, the nature of AC as detected by XPS appears to be extremely repetitive.’ The characteristic features found on the surface of most materials simply exposed to normal humidity, reasonably clean, STP air are all quite similar. This is particularly true for different silicates. Thus the C 1s spectra presented in Fig. 8(a) and (b) are quite typical. In this case one sees an overlay of the adventitious carbon spectra of two different samples of cummingtonite and a sample of hornblende. Although the characteristics are quite rough, close examination reveals certain repetitive patterns in addition to the principal C,H, peak assumed at 284.6 eV. There are, as expected, several weaker peaks upfield from the 284.6 eV peak and, although there are always obvious uncertainties in these peaks, they do suggest a pattern that repeats itself for all three systems. The rough positions for these peaks are marked off in Fig. 8 and, with assistance from B ~ z B , ’ ~ we have put together some general identifications in Table 7. As expected, there are several types of C-0 bonded systems identified, with the pattern seeming to emphasize the absorp- tion of carboxyl-containing systems and the effects of absorbed CO, plus perhaps adsorbed alcohols. Perhaps most importantly we find that there are noticeable gaps in the binding energy positions where the C-N-H (-285.6 eV) and O=C-N-H (-287.7 eV) species would be found. This observation is, of course, rein- forced by a lack of a significant N 1s peak. The signifi- cance of these observation will be described below.

The surface chemistry of murine cells. The principal reasons for the previous detailed consideration of adventitious carbon chemistry are to establish the form of analysis and to be able to differentiate the surface chemistry of the cells under study from the cell-free situations.

In order to achieve the latter we will consider a

Table 7. Organic binding energy ranges (in eV f 0.1) for the adventitious carbon on two cummingtonites and hornblende*

A B C D

1 1.2-1.6 C-O=C C-OH

2 2.2-2.5 C*=O Adsorbed 3 3.1 N H -C*=O Missing from adventitious

4 3.7-4.1 C-0-C*=O Adsorbed carbonyl 5 4.0-4.5 C 0 2 Absorbed

carbon

a Values given as positive shifts from 284.6 eV. A, peak label; B, binding energy range; C, organic bonding unit; D. specific identification and other comments.

XPS STUDY OF BIOCELLSILICATE INTERACTION 109

number of diverse, yet related, samples. The first group will be the cells alone. As one can see in Table 8, there are three samples that we purport consist of only cells. All were freeze-dried before XPS analysis. (We will comment below on the question of alive us. dead cells.) In addition, based upon the processing criteria common to bulk-oriented biochemistry, the solid material realized following the freeze-drying should consist of only cells, i.e. the MEM and 4% bovine serum in which the cells are suspended during culturing should be dissi- pated by the freeze-drying p roces~ .~ Unfortunately, as we now describe, this may not be the case for our outer surface-oriented results !

Before we consider the cell-only situation it will be instructive to examine the results for the case in which a silicate (cummingtonite) was treated with only the medium (MEM + 4% bovine serum) and thus put through all of the processing steps (without cells) followed by drying and examination by XPS.3 As noted above, the resulting silicate material exhibited many of the binding energies and quantitative characteristics of

Table 8. Select peak position binding energies and, in parenth- esis, linewidths (in eV) for freeze-dried cells”

Cells separated Cells separated Raw cells from cummingtonite from chrysotile

c 1s

0 Is

p 2P3P N Is

Na Is Ca 2p Si Al Fe M g

284.6 (1.67) 285.8 287.6

531.7 (3.0) 531.2 532.6 533.2

133.1 (1.8) 399.65 401.75 moderate 402.5 (1.8) 1071.2

Moderate 0 0 0 0

284.6 (1.75) 286.0 287.3 287.6 531.95 (3.0) 531.4 532.45 533.2

133.0 (2.1 ) 399.55 (2.0) 401.8 small

1070.9 Small

Very small 0 0

Small

284.6 (1.68) 285.8 287.6

531.4 (3.0) 530.1 9 532.4 532.4 533.0 132.9 (1.85) 399.5 (1.95) 401.5 small 402.4 1071.3

Very small Small 0 0

Very small

a Corresponding values in Table 7 and References 24 for design- ations of probable contributors.

unaltered cummingtonite, particularly in so far as the relative concentration of the lattice cations. Before one dismisses this example as an unaltered cummingtonite, however, the relative concentrations listed in Table 9 should be considered. In this case one should note that the sample in question exhibits a certain increase in nitrogen over untreated cummingtonite, as well as a growth in [C]/[Si]. Both of these results suggest that instead of being unaltered, the medium-treated cummingtonite has been surface coated during drying by an adsorbed layer of the medium. Close XPS examination reveals that (despite their extensive presence in the MEM) very little NaCl and NaHCO, are adsorbed onto the surface of the ~ummingtonite,~ but this is not surprising since these soluble salts should be removed during the washing step, but, on the other hand, the less-soluble organic species, e.g. glucose and certain amino acids such as l-gluatamine, may not be entirely removed during the distilled water washing. In addition, the principal blood components, such as hemoglobin, sugars and cholesterol, also may be adsorbed from the serum, along with some triglycerides and phospho- l i p i d ~ . ~ However, the latter should provide a distinct phosphorus peak structure in any XPS of the residue, and only a moderate amount is detected. The latter is an important point that we will return to later. Before we leave this consideration of the ‘residue’ apparently created by the medium dried onto cummingtonite, it will be instructive once again to utilize our previous results and the data of B&BZ4 to see if we can identify some of the chemistry of the medium residue. Based on the results for the C 1s spectrum presented in Tables 7-9, we suggest that a mixture of surface-oriented species containing C-N-H, C-OH, C*-0-C=O and NH-C=O are detected. These results are consistent with the adsorption of a mixture of glutamine, glucose, cholesterol and triglycerides.

We now return to an analysis of the separated cells. The three samples in question refer to one sample in which the murine cells were freeze-dried and examined by XPS, plus one each of freeze-dried cells extracted from cummingtonite and chrysotile. The relative quanti- fications for these samples are listed in Table 9, whereas the key resulting binding energies are presented in Table 8. In these three cases we have an advantage over the previous medium-only system in that no silicate with its resulting 0 1s and cations is present. The [N]/[C] ratios for the three cell-only samples are sur- prisingly reduced compared to the previously described

Table 9. CeP elemental concentration ratios for various systems based on organic units

Cells Cells separated from chrysotile Cells separated from cummingtonite Cells + cummingtonite Cells +chrysotile Cummingtonite Cummingtonite-separated cell Chrysotile-separated cell (2) Chrysotile-separated cell (1 ) Chrysotile-separated cell (3) Cummingtonite + medium Chrystile +medium

CNliCcl

0.1 0.085 0.08 0.34 0.24 0.31 0.29 0.1 5 0.1 3 0.074 0.31 0

COl/lSil

a, a, W

8.5 3.6 4.9 5.0 3.4 3.7

4.75 0

CNli[Ol

0.5 0.68 0.5 0.7 0.1 4 0.22 0.2 0.09 0.094 0.2 0.375 0

CPliCNl

0.236 0.255 0.36 0.1 2 0 Small 0 0 0 Small Small 0

CF’liCol

0.1 18 0.1 7 0.1 8 0.087 0 Small 0 0 0 Small Small 0

CCliCSil

a, a, a,

17.0 2.1 6 3.7 3.5 2.05 2.6

5.7 2.9

COl/CCI

0.2 0.1 3 0.1 6 0.47 1.7 1.3 1.4 1.8 1.4 0.34 0.85 2.1

110 T. L. BARR ET A L

medium-coated silicate case, but the [N]/[O] ratio is up, and there is a significant presence of phosphorus (see Table 9 and Fig. 9). The resulting C Is, 0 1s and N 1s binding energies establish the presence of C,H, , C-N-H and O=C-N type species [note Fig. 9(a)]. The evidence does not preclude C-OH type species, but substantial amounts of the latter are not indicated. In addition, there is ample proof of the presence of organic phosphate species [see Fig. 9(c)]. All of this is indicative of the detection of animals cells following the release (washing away) of the water and perhaps the lipids. The principal species detected are therefore proteins and a moderate amount of nucleic acids with their interconnecting phosphate bridges. The lack of presence of the O=C*-OR units of the lipid and the C*-OH species of the polysaccharides are not entirely understood, unless they also have been selectively washed away.

Before we unequivocally attribute the latter analysis to the detection of the surface chemistry of tumor cells, it is important to realize that our deposited cells may

n

also be coated upon freeze-drying by the medium, just like the previously described cummingtonite sample. There are certain characteristic differences between the resulting spectra of the cells and medium, however, suggesting that in the former case the surfaces of the freeze- dried cells are at least partially exposed to XPS detection.

Key XPS C, N, P and 0 results have also been devel- oped for cases in which the cells were freeze-dried directly to cummingtonite and chrysotile samples [see Tables 5 and 9 and Fig. 8(c)]. In these cases the concentrations and binding energies confirm that organic species and other components frozen from suspension are being detected, with the structure of the C 1s mani- folds and the [N]/[C] ratios similar to that produced by the medium on the cummingtonite sample. In the present cases, however, the variable presence of phosphorus and the resulting binding energies in the case of cummingtonite are indicative of the detection of cellular species rather than medium species. There is little doubt that the XPS analysis is detecting protein units,24 but

2 9 4 4 3 2 9 2 8 5 2 9 1 2 8 2 8 9 7 2 8 8 1 3 2 8 6 5 1 2 8 4 9 8 2 9 2 4 281 83 2 8 0 2 5 408 88 40651 404 55 401 79 29943 297 06 394 70

Binding Energy ( rV i Binding Energy (eV)

14096 13661 1 3 6 2 5 1 3 3 8 9 ' 3 1 5 3 1 2 9 1 6 1 2 6 3

Binding Energy (eV)

Figure 9. Representative C 1 s, N I s and P 2p spectra for freeze-dried untreated cells

111 XPS STUDY OF BIOCELL-SILICATE INTERACTION

the more diflicult role of defining the origins of these species remains unanswered.

Similar forms of organic ESCA analysis has been attempted on the silicate surfaces that were separated from cellular (medium) systems following extensive mixing. The resulting data are also reported in Table 5 and 9. Once again the presence of the significant N Is peaked at - 399.8 eV,24 and the character and positions of the C 1s peaks strongly suggest retention of significant depositions of organic units on the surface of the silicates. The lack of phosphorus and the C Is binding energies realized suggest the presence of substantial medium, along with the detection of cellular species. (Once again, a key feature is the obvious presence of substantial C-0-C*=O at -288.8 eVZ4).

It should be noted that the principal suppositions in the aforementioned analysis are also supported in the C, N and P analysis of the silicate-only systems (both chrysotile and cummingtonite). Thus, as previously noted, the C Is spectra for both of these materials are typical of air-exposed oxides subject to adventitious carbon. Furthermore, both the nitrogen and phosphorus intensities are essentially zero for both of these materials.

CONCLUSION

The most obvious feature of this research is its preliminary nature. We have made a number of novel observ- ations regarding the surfaces of silicates, particularly when interacting with biocellular materials, and many of these have obvious relevance for the eventual goal of providing useful information on the mechanistic description of the pathogenesis of select silicates; however, there still remain a number of general major problems areas that have not been approached.

Several of these problems were not truly appreciated when the work began. The most obvious is the tremen- dous complexity of the undertaking that results from the diversity of the materials involved. Thus, for example, we attached specific names to the geochemical units employed in each stage of the investigation, but the substantial range of compositions that are ‘umbrel- led’ under each designation (even within the same section of rock) substantially complicates this analysis, which depends so much on chemical differentiation. On the positive side, however, we can state emphatically that our initial goal of proving the utility of this type of marriage between, on the one hand, complex silicates and cellular materials, and, on the other, very specific surface analysis tools (particularly XPS) has been a substantial success. We have been able to detect the presence and behavior of the individual surfaces and follow them through a variety of complex process steps that should be reasonable simulations of the ‘real life’ experi- ences of these materials in nature. A central, unique feature of these studies is that they are the first to monitor not only the behavior of the surface of the cells but also the first to map the evolving chemistry of the silicate minerals.

In this regard, we have been able to: (1) detect and describe in some detail the surface

chemistries of a variety of types of natural silicates.

(2) in particular, we have provided the first detailed description of the chemistry of a number of serpentines and amphiboles.

(3) in this manner, we have shown how the bonding between the tetrahedral (silicate) and octahedral sublayers vary with composition and general structure.

(4) the particular influence of counter-cation types and also the variations with changes in the intra-lattice cations have also been pointed out.

(5) We have also begun to address the crucial question of identification of encapsulated byproducts and ex-solutions.

( 6 ) We have established that biocellular bodies (in this case, murine tumor cells) may be grown on some of these silicates and their surface involvement differ- entiated by XPS.

(7) The process chemistry has been shown to follow reasonable and generally consistent evolutionary steps during the cell growth and culture for both silicates and cells.

(8) Interactions between cells and the select silicates examined have been shown to alter the silicates.

(9) In the case of the amphibole cummingtonite, these alterations feature: the apparent selective surface ‘agglomeration’ (segregation?) of Fe, A1 and perhaps Mg; the erection of direct chemical bonds between cells (medium) and silicates involving the silicon-oxygen units, thus Si-0-C bonds are detected; the conversion of the silicate contained aluminum (originally detected in both tetrahedral and octahedral sublattices) to either tetrahedral- only positions and/or the creation of A1,0,.

(10) We have also been able to monitor certain aspects of the chemistry of the organic units during these processes, which in some cases seem to be due to the presence of the murine cells, but we are presently also aware of the persistent XPS detection of the medium in which the cells are originally emersed.

FUTURE RESEARCH

A number of questions has not yet been successfully addressed in these studies and warrant future consideration:

During the various treatments and analysis stages, biochemists are interested in whether the cells are alive or dead? We have some suggestions in this area regarding our studies (e.g. suspended ani- mation !) but must address the question with more care. We need to develop protocols for differentiating cell from medium effects, i.e. enhance our separation science. This is a difficult proposition for outer surface science in the 0 50 A regime. We have addressed some aspects of the geochemistry us. biochemistry question, finding that the selective surface ‘migration’ of an interaction with lattice cations is easier than the former group may have recognized, but we still doubt the contention of certain groups of biochemists who seem to feel that the mere grinding of silicates in a mortar will


somehow break M - 0 bonds (e.g. fracture an amphibole across one of its ‘I beams’).25 The amount of energy generated in the grinding process (equivalent, at most, to that of van der Waals bonds) will obviously only fracture solids along structural anomalies, e.g. twinning planes or other stacking faults. Thus, with respect to amphiboles, these features are concentrated in the zigzag regions occupied by the counter-cation between the I beams. ’

(4) We must now expand our efforts in two key areas: the involvement of more types of silicates, including some that seem to be biologically inactive; and the inclusion in our protocols of changes in conditions (e.g. temperature) to try to simulate environmental effects.

( 5 ) Most important, we must recognize that little in our present effort addresses the crucial question of pathogenesis from fibers us. Fe. Until this is examined in a more scientific manner, we will be unable to establish concrete steps towards a mechanism for silicate pathogenesis.

but still often questioned and should not be blindly accepted. Recently one of the authors published the first detailed critical examination of the origins and use of AC and, although defending the practice, acknowledges that far greater clarification is needed before anything approaching blanket acceptance is achieved. Second, the authors have relied on the extensive history in our group of the successful use of XPS to analyze the surface chemistry of silicates.’ This form of analysis is now in use in dozens of laboratories throughout the world,22 but, as recently emphasized by one of us (T.L.B.), it is a form of surface analysis and significant (sometimes undetected) variations with bulk behavior may result. This is an important feature in the present program and one of the principal reasons for the inclusion of other forms of analysis, such as MAS-NMR.26 Third, at present, XPS must be viewed as the ‘best’ (because it is the only) form of surface analysis of the interactive questions raised in the present study. Other methods of surface analysis, such as SIMS and AES, that may be superior to XPS in certain key areas are presently under study.

General statement

Several additional features critical to this paper need to be emphasized’ use is made throughout of adventitious carbon (AC) as a method for establishing the binding energy scale. This method is mature

Acknowledgements

Helpful discussions with Professor J. Klinowski, Dr H. H e and Dr P. Evans of the University of Cambridge, UK are gratefully acknow- ledged.

REFERENCES

1. R. C. Brown J. A. Hoskins and N. F. Johnson (eds), Mecha- nisms in Fibre Carcinogenesis. NATO AS1 Series A, Vol. 223, p. 387. Plenum Press, New York (1 991).

2. D. W. Kamp, P. Graceffa, W. A. Pryor and S . A. Weitzman, Free Radical Biol. Med. 12, 293 (1 992).

3. S. Seal, S. Krezoski, S. E. Hardcastle, T. L. Barr, D. H. Petering, C. F. Cheng, J. Klinowski and P. H. Evans, J . Vac. Sci. Technol. A1 3, 1 260 (1 995).

4. R. Ian Freshney, Culture ofAnimal Cells, 2nd Edn. Wiley-Liss, New York (1 987).

5. Sigma Cell Culture Reagents. Sigma Chemical Co., MO, USA (1988).

6. T. L. Barr and S. Seal, J. Vac. Sci. Technol., A13, 1239 (1995).

7. T. L. Barr and M. A. Lishka, J. Am. Chem. SOC. 108, 3178 (1986).

8. T. L. Barr, Modern ESCA. CRC Press, Boca Raton, FL (1994). 9. T. L. Barr, J. Vac. Sci. Technol. A9, 1793 (1991).

10. T. L. Barr, Crit. Rev. Anal, Chem. 22, 229 (1 991 ). 1 1 . W. A. Deer, R. A. Howie and J. Zussman, An Introduction to

the Rock-Forming Minerals, 2nd Edn. Longman Scientific and Technical Press, London (1 992).

12. T. L. Barr, Appl. Surface Sci. 15, 1 (1 983); T. L. Barr, Zeolite 10, 760 (1990); T. L. Barr, L. M. Chen, M. Mohsenian and M. A. Lishka,J.Am. Chem. SOC. 110,7962 (1988).

13. T. L. Barr, Microporous Mater. 3, 557 (1 995). 14. B. Herreros, H. He, T. L. Barr and J. Klinowski, J. Phys. Chem.

15. T. L. Barr, S. Seal, H. He and J. Klinowski, Vacuum 46, 1391

16. H. He, T. L. Barr and J. Klinowski, Clay Miner., 30, 201

17. S. Seal, S. Krezoski, D. Petering, T. L. Barr. J. Klinowski and P.

18. W. E. Ernst,Amphiboles. Springer-Verlag, New York (1 968). 19. H. W. Jaffe, Crystal Chemistry and Refractivity. Cambridge

20. T. L. Barr, Crit. Rev. Anal. Chem. 22, 1 13 (1 991 ). 21. T. L. Barr, in Practical Surface Analysis, 2nd Edn, ed. by D.

Briggs and M. P. Seah, Chapt. 8. Wiley, Chichester (1 990). 22. D. Briggs, in Practical Surface Analysis, 2nd Edn, ed. by D.

Briggs and M. P. Seah, Chapt. 9. Wiley, Chichester (1 990).

98, 1302 (1 994).

(1 995).

(1995).

Evans, submitted toJ . Vac. Sci Technol. (1 995).

University Press, Cambridge (1 988).

23. G. Barth, R . Linder and C. Bryson, Surf. interface Anal. 11, 307 (1 988).

24. G. Beamson and D. Briggs, High Resolution XPS of Organic Polymers. Wiley, Chichester (1 992).

25. V. Castranouve, V. Vallyathan, K. Vandyke and N. S . Dalal, in Effects of Mineral Dust on Cells, NATO AS1 Series, Vol. 11 30, p. 181. Plenum Press, New York (1 989).

26. J. Klinowski, Annu. Rev. Mater. Sci. 18, 189 (1 988).

x-ray photoelectron spectroscopic study of the possible interactions of biocells with the surfaces...

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