This action might not be possible to undo. Are you sure you want to continue?
Imaging the Action of Fluid Flow Blocking Agents on Dentinal Surfaces Using a Scanning Electrochemical Microscope
Julie V. Macpherson, Mark A. Beeston, and Patrick R. Unwin"
Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK
Nigel P. Hughes and David Littlewood
Unilever Research Port Sunlight Laboratory, Quarry Road East, Bebington, Wirral L63 3JW, UK Received May 15, 1995. In Final Form: July 24, 1995@
Scanningelectrochemicalmicroscopy (SECM)is used in the constant height imaging mode to characterize fluid flow through a porous dentine slice, subjected to pressures similar to pulpal pressures measured in vivo, and to assess the effectivenessof calcium oxalate ds a blocking agent of dentinal tubules. Local fluid flow rates and the topography of the dentine surface are mapped by monitoring the transport-limited current for the oxidation of ferrocyanide mediator at a tip ultramicroelectrode,scanned in close proximity to a target area of the surface, with and without an applied pressure across the slice. Following the applicationof the blocking agent, calcium oxalate, to the surface, subsequent scans in the same area reveal that all of the dentinal tubules are occluded and solution flow is inhibited. By converting diffusion-limited current images measured in the absence of pressure, before and after the application of the blocking agent, to corresponding topographical maps, it is demonstrated that the thickness of the precipitated calcium oxalate layer covering the surface can be estimated.
Introduction have used SECM to investigate the primary routes ofionic transport through skin during iontophoresis. In these Scanning electrochemical microscopy (SECM)1-3has studies the localized diffusional fluxes of electroactive been shown to be a powerful tool for imaging both the species through individual pores in the skin membrane t ~ p o g r a p h yand~the reactivity of solidlliquid interfaces, ~! were measured. with micrometer and submicrometer resolution. In the In recent work, we have further developed this approach latter area, SECM has been used to study a wide range and shown how SECM can be used to characterize and of interfacial processes a t the local level, including electron quantify local convective flow through dentine slices on t r a n ~ f e renzymatic a ~ t i v i t y , ~ , ~ ,~ etching,8 c o r r o ~ i o nand ,~ the micrometer scale, following application of a pressure dissolution kinetics.10-12 SECM has also been used typical of pulpal pressures measured in These successfully to determine local mass transfer across studies provided the first demonstration that fluid flow interfaces.13-16 In particular, White and c o - ~ o r k e r s l ~ - ~ ~ rates across dentine were spatially nonuniform, in accordance with ex: situ ultrastructural and clinical studies, Abstract published in Advance A C S Abstracts, September 15, which has shown that dentinal tubules may be completely 1995. or partially blocked with mineral aggregates or organic (1) Bard, A. J.;Fan, F.-R. F.; Kwak, J.; Lev, 0. Anal. Chem. 1989, 61. 132. solids.ls - - , ~-~ (2) Kwak, J.;Bard, A.J. Anal. Chem. 1989,61, 1221. Quantitative measurements of dentinal fluid flow rates, (3)Bard,A.J.;Fan,F.F.;Pierce,D.;Unwin,P.R.;Wipf,D.O.;Zhou, both in vitro and in vivo are of considerable importance F. Science, 1991,254, 68. in the development of new strategies for the treatment of Lee, C.; Kwak, J.; Bard, A. J.; Proc. Natl. Acad. Sci. 1990,87, (4) 1740. dentinal hypersensitivity. This condition arises following (5) Wipf, D. 0.;Bard, A. J. J. Electrochem. Soc. 1991,138,469. (a) the exposure of dentine, a calcareous material sandwiched (b) Bard, A. J.; Mirkin, M. V.; Unwin, P. R.; Wipf, D. 0.J. Phys. Chem. between the pulp and enamel which contains tubules, 1992,96,1861.( c ) Mirkin, M. V.; Richards, T. C.; Bard, A.J. J. Phys. Chem. 1993,97, 7672.(d) Engstrom, R.C.; Small, B.; Katton, L. Anal. typically 1-2 pm in diameter, at a density of ca. 106-107 Chem. 1992,64,241. cm-2. In response to tactile, thermal, or osmotic stimuli, (6) Pierce, D.T.; Unwin, P. R.; Bard, A.J. Anal. Chem. 1992,64, (a) dentinal fluid can flow outward through exposed tubules (b) Pierce, D. T.; Bard, A. J . Anal. Chem. 1993,65, 3598.(c) 1795. Horrocks, B. R.; Schmidtke, D.; Heller, A,; Bard, A. J . Anal. Chem. into the oral ~ a v i t y . Simultaneously, toxins can diffuse ~~-~~ 1993,65,3605. along a concentration gradient to the pulp,24 causing (7) Shiku, H.; Takedo, T.; Yamada, H.; Matsue, T.; Uchida, U. Anal. inflammation and eventual pulp death. This overall Chem. 1996,67,312. movement offluid is thought to initiate a mechanoreceptor ( 8 ) (a)Mandler, D.; Bard, A.J. J. Electrochem. Soc. 1990,137,2468; (b) Mandler, D.; Bard, A. J. Langmuir, 1990,6 , 1489;(c) Meltzer, S.; response in the pulpal nerve fibres which causes a Mandler, D. J. Chem. Soc. Faraday Trans. 1995,91,1019. sensation, detected as pain.25 (9) Wipf, D. 0. Colloids Surf A. 1994,93,251.(b) Casillas, N.; (a)
Charlebois, S. J.;Smyrl, W. H.; White, H. S. J . Electrochem. Soc. 1994,
(10) Macpherson, J.V.; Unwin, P. R. J . Phys. Chem. 1994,98,1704. Macpherson, J.V.;Unwin,P.R.J. Phys. Chem. 1994,98,11764. (11) Macpherson, J. V.; Unwin, P. R. J. Phys. Chem. 1996,99,3338. (12) Macpherson, J. V.; Beeston, M. A,; Unwin, P. R. J. Chem. Soc. (13) Faraday Trans. 1996,91,899. (14) Scott, E.R.; White, H. S.; Phipps, J. B. J. Membr. Sci. 1991,58, 71. (15)Scott, E.R.; Laplaza, A. L.; White, H. S.; Phipps, J . B. Pharm. Res. 1993,10, 1699. (16) Scott, E. R.; White, H. S.; Phipps, J . B. Anal. Chem. 1993, (a) (b) J 65,1537. Scott, E. R.; Phipps, J. B.; White. H. S. . Invest. Dermatol. 1996,104,142.
(17) Macpherson, J. V.; Beeston, M. A,; Hughes, N. P.; Littlewood, D.; Unwin, P. R. J. Chem. Soc. Faraday Trans. 1996,91, 1407. (18) Thomas, H. F.; Payne, R. C. J . Dent. Res. 1983,62,532. (a) (b). Yoshiyama, M.; Noiri, Y.; Ozaki, K.; Uchida, A,; Isikawa, Y.; Ishida, H. J. Dent. Res. 1990,69,1293. Graf, H.; (c) Faddi, S.;Burgin, W. J . J . Oral 201. Rehab. 1990,17, (19) Pashley, D. Matthews, W. G.;Arch. Oral. Bid. 1993,38,577. H.; Griffths, H.; Morgan, G.; Williams, K.; Addy, M. J . Periodont. (20) Res. 1993,28, 60. (21) Matthews, B.; Vongsavan, N. Arch. Oral Biol. 1994,39,875. Van Der Graaf, E. R.; Ten Bosch, J . J.Arch. OraZBioZ. 1988,33, (22) 375. (23) Vongsavan, N.; Matthews, B. Arch. Oral Bid. 1992,37, 175.
0 1995 American Chemical Society
3960 Langmuir, Vol. 11, No. 10, 1995
One class of treatments for dentinal hypersensitivity is the application of blocking agents which occlude the open t ~ b u l e s . ~ ~ -blocking .agents are either applied in The ~ ~ the form of insoluble crystals,26y28 e.g., calcium oxalate, calcium hydroxide, resins, and adhesives, which act directly to block the tubules, or as a soluble salt solution,26 the ions of which react with soluble calcium or phosphate ions a t the dentine surface to form an insoluble layer, e.g., calcium oxalate may be formed through the application of a potassium oxalate solution. Previous in vitro studies of the effectiveness ofdentinal blocking agents have employed methodologies which are only able to measure the net permeability of the entire ample.^^-^^ Moreover, to our knowledge, the surface of dentine has not been imaged in situ following the application of a blocking agent. The aim of this paper is to demonstrate how SECM can be used to provide valuable in situ information on the efficacy of blocking agents through a series of simple, rapid experiments. First, local fluid flow rates and the topography of the dentine surface are mapped by monitoring the transport-limited current for the oxidation of ferrocyanide mediator at a tip ultramicroelectrode, scanned in close proximity to a target area of a dentine surface, supported as a slice, with and without an applied pressure. This allows active local permeable regions to be identified. Secondly, following the application of the model blocking agent, calcium oxalate, to the surface, subsequent scans in the same area, again with and without a n applied pressure, provide images which may be used to assess the effectiveness of the agent a t the local level. Finally, by converting diffusion-limited current images measured in the absence ofpressure, before and after the application of the blocking agent, to corresponding topographical maps, it is demonstrated that the thickness of the precipitated calcium oxalate layer formed on the surface can be estimated. Although the studies described herein are concerned with a topic in the dental field, the approach described should be generally applicable to similar types of processes in other areas of interfacial chemistry. For example, the determination of film or layer thicknesses is a general problem in surface chemistry. The studies in this paper demonstrate for the first time that SECM is a powerful technique for imaging the thickness of layers on surfaces on the micron and submicron scale.
Macpherson et al.
mol dm-3 potassium chloride (Fisons, Analytical Reagent) aqueous solution (Milli-Q reagent grade water) was used as the electrochemical mediator. Apparatus and Instrumentation. The SECM instrumentation employed has been described in detail e l s e ~ h e r e . ~ ~ - ~ ~ % ~ ~ The ultramicroelectrode (UME) tip was a 25 pm diameter Pt disk electrode. Although the size of this probe precluded resolution at the individual tubule level, it allowed images to be obtained over large areas of a sample. The UME acted as the working electrode in a conventional two-electrode setup, with a silver wire serving a s a quasi reference electrode (AgQRE). The prepared dentine slice was sealed, flush in the end of a 4 mm outer diameter glass tube, using a “Superglue”/varnish mixture (Bostik, Leicester, UK). The tube was then mounted vertically in the base of an SECM cell, as reported for similar ~ o r k ,so ~ , ~ ~ ~ that the flat dentine surface lay perpendicular to the axis of the UME tip. The cell was filled with the mediator solution so that the solution level covered the surface of the dentine slice. A reservoir containing the electrochemical mediator solution was attached to the other end of the glass tube, uia poly(tetraflu0roethylene) tubing. The pressure across the dentine slice was readily controlled by varying the height between the solution level in the reservoir and that in the SECM cell. Procedure. Images of the transport-limited current for the one-electron oxidation of ferrocyanide ions, versus tip position, were obtained by scanning the tip a t 20 p m s-l, in a fixed plane above the dentine surface, typically at a n initial tip/dentine surface separation of 5 pm. This distance was set by carefully positioning the tip onto the dentine surface so that they contacted and then moving the electrode back from the surface the required distance. For all imaging experiments, the electrode potential was held a t 0.7 V versus AgQRE. All of the images presented in this paper relate to the same 400 p m x 400 pm area of a dentine surface, but are typical of the results obtained on several samples ofdentine. Images were recorded in the constant height mode, with the dentine slice subjected to the following sequentially imposed conditions. (i) No pressure, i.e. no dentinal fluid flow. (ii) 2 kPa of pressure (equivalent to a 20 cm head of solution), sufficient to activate fluid flow. This pressure is similar to values ofthe pulpal pressure (iii) measured in uivo.23,35 No pressure, following the application of the blocking agent to the surface. (iv) 2 kPa of pressure with the blocking agent on the surface. Application of the blocking agent to the surface of dentine between scans ii and iii was effected by first moving the tip far away from the surface, specifically 500 pm in the z direction (normal to the surface) and 3000 p m in the x direction (parallel to the surface), and draining the cell of solution. Solutions ofthe reagents calcium chloride and potassium oxalate were then pipetted onto the surface in microliter quantities. After allowing 5 min for the precipitation ofcalcium oxalate to reach completion, the surface was carefully washed with Milli-Q reagent water to remove any excess reagents and the cell was again drained. The cell was refilled with the mediator solution and the electrode was registered in its original position. The ability to move and reposition the electrode with the original z co-ordinate with high precision, in order to allow the application of the blocking agent, is ofvital importance since, for a tip scanned in a constant plane, the difference in the topographical (no pressure) images obtained with and without the blocking agent, provide information on the thickness of the calcium oxalate layer, as described below. Although the SECM e m p l ~ y e d l ~has ~ , ~ ~ - ~ integrated optical encoders which provide a resolution in tip position of 0.05 pm, we evaluated the repositioning capabilities of the SECM system in a separate experiment as follows. The steady-state current response of the tip placed 5 p m away from the dentine surface was compared the value obtained after the tip had been moved 500 pm in the z direction and 3000pm in thex direction, the solution was drained from the cell, fresh solution was added to the cell, and the tip was returned to the original co-ordinates. The variance in the measured currents was less than 2%, indicating that the tip (34) Macpherson, J . V.; Marcar, S.; Unwin, P. R. Anal. Chem. 1994,
Materials and Solutions. The dentine slice used was ca. 50 pm thick and taken from a coronal section cut from the outer third of dentine of y-irradiated human erupted premolars. The organic smear laye133 which usually coats the surface of the dentine, and can partially occlude tubules, was removed by immersing the dentine in 3 mol dm-3 citric acid for 10 s and then thoroughly rinsing in Milli-Q reagent water. Electron microscopy revealed a mean tubule density of ca. 1 x lo7 while tubule diameters were ca. 2 pm. The model dentinal blocking agent, calcium oxalate, was prepared from the precipitation reaction between 0.1 mol dmV3 calcium chloride and 0.1 mol dm-3 potassium oxalate. Potassium ferrocyanide trihydrate (Fisons, Analytical Reagent) at a concentration of 0.01 mol dm-3 in a 0.5
(24) Vongsavan, N.; Matthews, B. Arch. Oral BioZ. 1991,36,641. (25) Brannstrom, M.; Johnson, G. Acta Odont Scand. 1970,28,59. (26) Addy, M.; Dowell, P. J. CZin. Periodontol. 1983,10, 351. (27) Dowell, P.; Addy, M. J . CZin. Periodontol. 1983,10, 341. (28) Pashley, D. H.; Leibach, J. F.; Homer, J. A.J. PeriodontoZ. 1987,
58,19. (29) Greenhill, J. D.; Pashley, D. H. J. Dent. Res. 1981, 160, 686. (30) Pashley, D. H. J. Endodontol. 1986,12, 465. (31) Pashley, D. H.; O’Meara, J. A,; Kepler, E. E. J. Periodontol. 1984,55, 522. (32) Addy, M.; Mostafa, P. J. Oral Rehab. 1989,16, 35. (33) Dippel, H. W.; Borggreven, J. M. P. M.; Hoppenbrouwers, P. M. M. J. Prosthet. Dent. 1984,52, 657.
(35) Andrews, S.A,; Van Hassel, H. J.; Brown, A. C. J . Hosp. Dent. Practice 1972,6,49.
Scanning Electrochemical Microscope Imaging
SECM tip scanned in fixed x,y plane
ox Red Red
Langmuir, Vol. 11, No. 10, 1995 3961
x scan I pm
;h c n a ; topography
increased curr nt due to fluid flow thro gh tubules 0.8
0.5 i I i (a)
Figure 1. Schematic (not to scale) illustrating how SECM images the surface topography of, and convection across, a porous dentine sample via changes in the transport-limited current response of a UME tip.
Figure 3. Transport-limitedcurrent imagesof the area imaged for Figure 2, after treatment ofthe surfacewith calcium oxalate. Images were recorded (a)with no pressure across the slice and (b) under a fluid pressure of 2 kPa.
Figure 2. Transport-limited current images recorded over a 400 pm x 400 pm area of dentine (a)with no pressure applied across the dentine slice and (b) with a fluid pressure of 2 P a
across the slice. could be returned to its original position without the introduction of any significant errors from piezoelectric hysteresis and drift.
Results and Discussion The principles behind the use of SECM to image the topography of, and mass transport across, a porous substrate are reviewed schematically in Figure 1. When the UME tip is held at a potential for the transport-limited oxidation of the ferrocyanide mediator (denoted by Red in Figure 1)and scanned in close proximity to the dentine surface (such that the initial tiphubstrate distance is typically less than the radius of the UME), the current flowing a t the electrode is governed by the rate of local mass transport (convection and diffusion). Under conditions where there is either no pressure applied across the dentine slice or the tip sits above an area of the surface where the tubules are blocked, transport of the mediator to the tip is by diffusion alone. In this situation, the magnitude of the current is inversely related to the tip to surface distance,2 and it is well-established that the current response can be used to provide a topographical map of the s ~ r f a c e In contrast, as the tip is moved over .~~~ an open tubule in the dentine slice, which has a pressure applied across it to activate fluid flow, mass transport of the mediator to the electrode by forced convection results in an increase in the current. By sequentially mapping the transport-limited current over an area of the surface, with and without an applied pressure, it is thus possible to image the topography of the surface and identify active permeable regions of the sample. A typical three-dimensional SECM image of the dentine surface when no pressure is applied across the slice is shown in Figure 2a. The current, i , detected as a function of tip position, has been normalized with respect to the
3962 Langmuir, Vol. 11, No.10, 1995
Macpherson et al.
treated dentine surface, after SECM experiments.
Figure4 Scanningelectron micrograph of the calcium oxalate .
Thickness of oxalate layer I
Thickness of oxalate layer I pm
F T '
y scan I pm
, 3 '2 1 0
dentine x scan I
-- 100 150
I C U R R E N T E
dp - d, = thickness of oxalate layer Figure 5 Schematic illustrating the current response when .
before (the lower, lighter image),and after (the higher, darker image)treatment with calcium oxalate. Images were produced by convertingthe diffusion-limited current data in Figures 2a and 3a to the corresponding tiphubstrate separations, via eq 1.(b)The calculated variation in the thickness of the calcium oxalate layer formed on the dentine surface. sample is nonuniform, as established previously for typical dentine mate15als.l~ The diffusion-limited current image recorded in the same area with no pressure across the slice, following the application of the blocking agent, is shown in Figure 3a. It can be seen that the i/i(-> values recorded are lower than those determined under identical circumstancesprior to the addition of the blocking agent (Figure 2a). Given that the tip was scanned in a constant plane to record both images, the reduced i/i(-> values can be attributed to a decreased distance between the tip and the surface, resulting from the formation of a calcium oxalate layer, offinite thickness, on the dentine surface, as demonstrated below. The variation of the transport-limited current with tip position recorded in the same area, with ca. 2 kPa of pressure applied across the calcium oxalate treated surface, is shown in Figure 3b. It is clear that parts a and b of Figure 3 are essentially identical, indicating that, in this area, the calcium oxalate treatment has successfully rendered all of the previously open tubules inactive to flow. The success of calcium oxalate in blocking flow across the dentine surface is readily explained by reference to Figure 4, an electron micrograph of a typical area of the surface taken after all of the SECM measurements had been completed. This clearly shows that calcium oxalate crystals form a dense layer on the surface. The thickness of this layer can be estimated from the in situ SECM data in Figures 2a and 3a. As shown schematically in Figure
(36) Mirkin, M. V.; Fan, F. -R. F.; Bard, A. J. J. Electroanal. Chem. 1992,328, 47.
Figure 6 (a) Topographical images of the surface of dentine .
the SECM tip is scanned in a constant plane over (i) the untreated dentine surface and (ii)the calcium oxalate treated dentine surface. In both cases the current, measured as a functionof electrodeposition in the x-y plane can be converted to a tipkubstrate position which reveals the topography of the surface. Subtractionof the tipkalcium oxalatesurfacedistances from the tip/dentine surface distances yields the variation in thickness of the calcium oxalate layer. steady-state diffusion-limited current which flowed when the electrode was positioned a long distance (at least 10 electrode diameters2)away from the surface, i(-). Under the conditions of this experiment, i depends only on the tip/surface distance and the data can be used to construct a semiquantitative map of the surface topography, as discussed below. In contrast, Figure 2b shows the same area of the dentine sample, but now with 2 kPa of pressure applied across the slice. The striking difference in the magnitudes of the currents in the two images can be attributed to fluid flow through dentine tubules, enhancing mass transport of the ferrocyanide mediator to the tip. The wide spatial variation in' the current enhancements observed by comparing parts a and b of Figure 2 indicates that the flow rate distribution across this area of the
Scanning Electrochemical Microscope Imaging 5, when the tip is scanned in a constant plane, with no pressure across the slice, the tip current response before the application of the blocking agent is governed by the tipidentine separation, while after the blocking agent has been added, the current depends on the tiplcalcium oxalate separation. By quantitatively relating the measured currents to tipisample separation in the two cases, the topography of the calcium oxalate layer may be revealed. To convert the diffusion-limited current images in Figures 2a and 3a to topographical maps of the surface, we used eq 1,36 which relates the negative feedback current to the tipisample separation:
Langmuir, Vol. 11, No. 10, 1995 3963
calcium oxalate layer thickness is in the range 0-10 pm, but is highly nonuniform.
SECM has been shown to be a useful technique for imaging the topography of dentine surfaces, identifying active permeable regions through which fluid flows under a small pressure, and for investigating the mode of action of calcium oxalate, a model tubule blocking agent. By comparing sequentially recorded transport-limited current images of a permeable area of dentine, which was subsequently treated with a blocking agent, it has been shown that calcium oxalate successfully occludes all dentinal tubules and inhibits flow. Moreover, by imaging the untreated and treated surface in the negative feedback mode a t constant height, the topography and thickness of the calcium oxalate layer formed on the dentine surface is readily estimated. Although we have focused on a n important topic in the dental field for this paper, it should be noted that SECM could find application in the measurement of local fluid flow rates in other microscopic systems. Additionally, the technique is generally well suited for in situ measurements of the thickness and topography of films or layers formed at solid/liquid interfaces.
+ 1.515IL+ 0.655 e~p(-2.4035/L)I-~
where L = d l a ,with d denoting the tipisurface distance and a representing the electrode radius. Strictly, eq 1 applies to a tip positioned above a n inert planar surface, but we recently demonstrated that it may provide semiquantitative topographical information for more irregular surfaces.12 Figure 6a is a composite plot of the variation of the tiphubstrate distance with tip position for the dentine surface before and after treatment with calcium oxalate. The z data have been plotted as -d to highlight the topographies of the two surfaces. The difference in the two data sets, shown in Figure 6b, effectively represents the thickness of the calcium oxalate layer in the x-y area scanned by the probe. This clearly demonstrates that the
Acknowledgment. We thank the EPSRC for supporting this work through a n equipment grant (GW H63739) and a n earmarked studentship for J.V.M. An EPSRCAJnilever CASE studentship for M.A.B. is gratefully acknowledged.
This action might not be possible to undo. Are you sure you want to continue?
We've moved you to where you read on your other device.
Get the full title to continue listening from where you left off, or restart the preview.