Anal. Chem. 1993, 65, 2288-2293
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Surface Analysis and Characterization of Chemically Modified Chalcogenide Fibers K.Taga,' D. Schwarzbach, G.Stingeder, M.Grasserbauer, and R. Kellner University of Technology Vienna, Getreidemarkt 911.51, A-1060 Vienna, Austria
I n this paper, the use of several analytical methods for the determination of the fiber surface coverage after each step of chemical immobilization of a n enzyme is reported. The enzyme glucose oxidase was immobilized on the surface of a n infrared transparent chalcogenide fiber using (3-aminopropy1)triethoxysilane and glutaraldehyde or (yglycidoxypropy1)triethoxysilane as a carrier as described in our previous papers. This reactive enzyme layer coating the core of the fiber serves to catalyze chemical reactions specifically. However, efficient chemical modification for the development of chemical sensors requires that the coating covering the fiber remains homogeneous and stable along the surface. I n order to investigate the surface and to control the quality (homogeneity and thickness) of such thin organic layers, it is necessary to exploit several surface analysis techniques. Secondary ion mass spectrometry was applied to prove the presence of oxygen and OH groups on the fiber's surface. Infrared spectroscopy and infrared microscopy were used to monitor the spectral changes to evaluate information about coatings on a molecular basis whereas the scanning electron microscope coupled to an energy-dispersive spectrometer was applied to evaluate information on a n elemental basis. Finally, atomic force miscroscopy was used to examine the morphology of the modified fiber surface. I n conclusion, the combined evaluation has shown that a n enhancement of the surface density of the active layer at the IRtransparent fibers is necessary to exploit the compound-specific potential of the new sensor.
INTRODUCTION Fiber-optic chemical IR sensors are complex new analytical systems, whose performance is strongly dependent on the quality of the thin chemically reactive layer around the active part of the light guide, where the specific or selective interaction between the sample and the system takes place. The eurface homogeneity affected by the multiple chemical modifications is determinant to the sensor functioning. The layers composing the reagent phase have to be dense and fully covering the active part of the sensor. Thus, surface investigation techniques have to be used on the one hand to understand all physicochemical processes taking place on the surface during the construction of the sensor and, on the other hand, to analyze and compare the derivatized surfaces on a microscopic and nanoscopic level with the different reagent carriers. These surface investigationsperformed with
* Author to whom correspondence should be addressed. 0003-2700/93/0365-2288$04.00/0
severaltechniques to satisfy all requirements needed to control such a complex product as a sensor will be described separately in this paper. Each technique has complementary capabilities and distinct objectives. Although the deposition of silanes as a coupling agent on inorganic material has been used and developed for many years's2 the investigation and the surface analysis of silanes on silicon oxide is still of great interest due to the complexity of the conformation and structure of the silane a t interfaces often resulting in multilayer and irregular surface morphol0gy.3
EXPERIMENTAL SECTION Chalcogenide fibers composed of AszsSeMTe17 having a diameter of 500pm were used for the chemical derivatization. These fibersbelong to a new generation of materials transparent in the mid-infrared (mid-IR) spectral range and are therefore suitable for the development of infrared chemical sensors. The chemical derivatization of chalcogenide fibers has been described previ0usly.'9~ Basically, the glucose oxidase (GOx) was immobilized on the fiber surfaces via (3-aminopropy1)triethoxysilane(3-APTS) and glutaraldehyde (GA) or via the (y-glycidoxypropy1)triethoxysilane (GPTS)as shown inFigure 1. The silanizationprocedures as well as the immobilization procedures are conventional modification techniques and have been presented in many applicationswhere proteins were bound to inorganic surfaces."'
SURFACE ANALYSIS INVESTIGATIONS Secondary Ion Mass Spectrometry (SIMS). For the development of chemical sensors, a very important question related to the derivatization of the fiber was the oxygen distribution on its surfaces. In order to generate chemisorbed silane layers on surfaces, OH groups are needed at the carrier surface (see Figure 1). The presence of OH groups on semiconductors, Le., on Si and Ge surfaces, is known. In addition, it is known that tellurium contained in chalcogenide fibers has a tendency to oxidize at room temperature to yield TeOz at the surface, which has absorption peaks at 12.98 and 14.92 pm in the IR spectral range.* However, no paper presents the oxygen distribution on particularly chalcogenide materials, which had therefore to be characterized with appropriate methods. This was done by dynamic secondary ion mass spectrometry (SIMS). This technique has a high sensitivity and selectivity which allows trace element analysis (1) Ishida, H. Polym. Compos. 1984, 5, 101. (2) Chiang, C.; Ishida, H.; Koenig, J. L. J . Colloid Interface Sci. 1980,
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(3) Vandenberg, E. T.; Bertilson, L.; Liedberg, B.; Uvdal, K.; Erlandsson, R.; Elwing, H.; Lundstrdm, I. J. Colloid Interface Sci. 1991, 147, 103. (4) Taga, K.; Weger, S.; Gdbel, R.; Kellner, R. Sens. Actuators B, in press. (5)Taga,K.; Kellner,R. SPIEProc. of theBthF"I'SConference,LBbeck, 1991. (6)Plueddemann, E. P. Silane Coupling Agents; Pleunum Press: New York, 1982. (7) Haller, W. In Solid Phase Biochemistry; Scouten, W. H., Ed.; J. Wiley & Sons: New York, 1983. (8) Haruvi-Busnach, I.; Dror, J.; Croitoru, N. J. Mater. Res. 1990,5 (6). 0 1993 American Chemical Society
ANALYTICAL CHEMISTRY, VOL 65. NO. 17, SEPTEMBER 1, 1993
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Two reaction schemes forthe covalent immobilization of an enzyme on a Chalcogenide fiber surfacevia 3-APTS (left)and GPTS (right). Table I. Operating Parameters Used for the Dynamic and
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Time [minutes1 Flgure 2. (a, top) leg-secondary Ion image of a cross-sectional chalcogenide fiber face (scan is 500 X 500 pm2). (b. bottom) SIMS depth profile of a freshly cleaved chalcogenide fiber face.
the ' 6 0 - image of a chalcogenide fiber cross section taken by aPhi-Atomika65WSIMS instrument. From thedepth profie of the rough fiber surface (Figure Zb), one can see that the oxygen concentration diminishes as a function of sputtering time whereas the elements contained in the matrix (core of the fiber) show constant intensities. This confirms that oxygen is enriched on the fiber surface and that the bulk is composed of homogeneous glass since the concentrations of As, Se, and Te remain constant within the noise level. The operating parameters used for the dynamic SIMS measurements are summarized in Tahle I. In addition, our interest was to find out whether or not hydroxide groups are responsible for silane bonding on the outmostfibersurface. TheSIMSimageand thedeptb profile showed only the oxygen distribution around the fiber, which isnotenoughtodeducethepresenceofOHgroups. Therefore, static time-of-flight secondary ion mass spectrometry (TOFSIMS) was used to gain additional information. In the last decade, it was shown that TOF-SIMS is a powerful technique for characterizing the chemical structure of the outermost surface of several material^.^ A detailed description of the TOF-SIMS instrument was presented in refs 10 and 11. Figure 3 shows a typical mass spectrum in the region of oxygen as obtained at different locations on the fiber surface. The operating conditions are
in microvolumes. The main part of the signal was generated from the first atomic layers. In the following investigation it was found that oxygen was indeed enrichedat thechalcogenidesurfaces. Figure 2ashows
(9) Lub,J.;vanVroonhoven.F. C. B. M.;vanLeyen,D.:Benninghov*~ A. Polymer 1988,29,998. (10) Bennighoven,A. J. Voc.Sei. Teehnol. 1985, A3 (3, 451. (11)Niehuis,E.;vanVelzen,O.N . T . ; L u b , J . ; H e l l e , , T . ; B e ~ h ~ n , A. Surf. Interface Anal. 1989, 14, 135.
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1, 1993
Wavenumbers in cm-' Flgure 4. ATR spectrum of GOx immobilized via 3-APTS and GA on the chalcogenide fiber surface (reference spectrum is the untreated fiber).
summarized in Table I. As can be seen1a significant OH- (at = 17 amu) signal could be detected at the surface, which is a strong indication of the reactivity of the chalcogenide fiber surface with 3-APTS or GPTS. Fourier Transform Infrared Spectroscopy (FTIR). Attenuated total reflection (ATR) FTIR spectroscopy is a widely used tool for the investigation of surface phenomena within the penetration depth of the IR radiation into the surrounding medium.12 During the fiber surface modification every single process was monitored in situ with the FTIR spectrometer. The ATR spectra were taken with a Bruker IFS-88 FTIR coupled to an MCT detector; 100 scans were coadded at a resolution of 4 cm-l. ATR spectra showing the progress of the silanization as a function of time and the activation procedure monitored with the FTIR spectrometer have already been presented in our previous investigation.13 However, the last step confirming the direct immobilization of the glucose oxidase to the fiber surface is shown in Figure 4, displaying the ATR spectrum of the reactive layer. The major IR bands of proteins can be assigned to the vibrations of the peptide linkage in the protein chain. A decrease of the C=O stretch band intensity compared to the intensity of the band before the immobilization indicates that the carboxyl groups have reacted with the enzyme. The band at 1663 cm-1 can be assigned to the amide I vibration of the enzyme.14Js The band at 1585 cm-l can be assigned to the amide I1 vibration, showing a smaller intensity than amide I. Fourier Transform Infrared Microscopy. FTIR microscopy provides ex-situ and nondestructive analysis. It requires very little or no sample preparation and gives the possibility of rapidly acquiring IR spectra of samples with a spatial resolution better than 40 pm. Small areas of interest can be easily manipulated and analyzed. For our investigation, a Bruker FTIR microscope equipped with an MCT detector was used to analyze the derivatized sample surfaces after each chemical process. Several spectra were recorded along the fiber axes at different locations in order to control the homogeneity of the derivatized surface (quality control). They were performed in the transmission mode with a constant aperture equal to 1.2 mm, corresponding to a focus diameter on the sample surface of approximately 90 pm.
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"Localized" spectra were taken along the fiber surface after each chemical derivatization process. A piece of fiber was positioned into the focus of the IR beam and afterward the interferogram signal was maximized using the motorized XY sample stage. Panels a and b of Figure 5 show typical spectra taken from a line scan of chalcogenide fibers on which glucose oxidase was immobilized on 3-APTS and GPTS. These results were unexpected in a sense that the sensitivity was high enough to get the spectral information at molecular level after every chemical treatment of the fiber. Since the reactive film on the fiber surface has a thickness up to 100 nm, and conventional IR microscopic measurements are done with film thicknesses varying from 10to 20 pm,lesuch high-quality IR spectra of the analyzed fiber were not expected at the beginning. An interpretation using a theoretical model has already been given in our earlier papers,"J* describing how the fiber geometry can dramatically enhance the sensitivity in the transmission mode of the FTIR microscope. Scanning Electron Microscopy (SEM). Surface topology and chemical composition measurements were made with a JEOL JSM-6400scanning electron microscope coupled to the Link EDS analyzer. This processing system was used to obtain spectra of the samples and to carry out quantitative analysis with the software package supplied from Link Analytical. Composition analysis was carried out with an energy-dispersive X-ray spectrometer (EDX) coupled to the scanningelectron microscope. The chalcogenidefiber samples (16) Katon, J. E.;Sommer, A. J.; Lang, P. L. Appl. Spectrosc. Reu.
(12) Harrick, N. J. Internal reflectance spectroscopy; Interscience: New York, 1967. (13) Taga, K.; Weigel, C.; Kellner, R. Vib. Spectrosc. 1990,1,125. (14) Susi, H.;Timaeheff, S. N.; Steven, L. J. Mol. Biol. 1968,37,231. (16)Henog, W.Angew. Chem., Int. Ed. Engl. 1987,26, 5.
1989,25, 173.
(17) Kellner, R.; Mizaikoff, B.; Taga, K.; Theiss, W.; Grosse, P. Fresenius. J. Anal. Chern., in press. (18) Mizaikoff, B.; Taga, K.; Kellner, R.; Toward theoretical limits of FTIR microscopy for ultra thin organiclayers. Appl. Spectrosc., in press.
ANALYTICAL CHEMISTRY, VOL. 65, NO. 17, SEPTEMBER 1. 1993
Table 11. Operation Parameters for Quantitative SEM Analysis with the EDX-ray Detector accelerating voltage (kV) current (A) spectralrange (keV) dead time ( % I live time (8) electron focus length (mm) magnification analysis mode
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silica-based materials such as silica gel or conventional glass.3 Thus EDX-ray analyses of the silane layers were not possible because of the coexcitation of the matrix. In the case of chalcogenide fiber materials, no X-ray emission from the matrix overlaps the silicon peaks. Therefore this sensitive method could he applied. X-ray spectra were recorded at different locations on each fiber and the intensity of the characteristic silicon Ka peak (at 1.743 keV) was measured. The spectra confirm the polymerization in island structures on the surfaces. As can he seen in Figure 7, the silicon X-ray intensityvariesfrom afew hundred toafewthousand counts, indicating the presence of different amounts of siloxanes. Atomic Force Microscopy (AFM). Biological applications of atomic force microscopy, though technically challenging, are destinated to he of great importance. Already, the AFM has imaged biological molecules such as amino acids or hipolymer macromoleculessuch as proteins and has showed the ability to measure three-dimensional profiles with nanometer resolution.20 Preliminary images of chalcogenide fibers chemically modified with APTS glutaraldehyde and glucoseoxidase were obtained with a Park Scientific atomic force microscope. Imaging was performed under air in a constant force mode using a Si3Nacantilever tip, resulting in a constant force in the order of l0-8-llF N. Parts a and h of Figure 8 show the topography and the distribution in the nanoscopic range of two typical reactive layers of the fiber surface. One can see that molecule aggregates are bound to the area and that, depending on the surface probed, the degree of coverage changes drastically.
DISCUSSION
Flgum 0. (a. top) SEM micrograph of a chalcogenide fiber derhratlzed whh APTS. (b. bottom) Magnification of (a).
have not been coated with gold for topographic investigations or for X-ray spectra collection. This preparation step could he omitted due to the good conductivity properties of the materials under the operating conditionssummarized in Table 11. The fiber samples were cut into pieces of 3-4-cm length and then mounted in a circular metal support appropriate for the sample holder of the instrument. The morphology of the derivatized fiber surfaces was examined in the secondary electrons emission mode. This surface analytical method has already been applied to the characterization of the electrochemical sensors.19 From the following SEM micrographs (Figure 6a,h), representing the reacted 3-APTS on the chalcogenide fiber surface, a heterogeneoussilane coverage can he seen. The photos reveal that, in all cases, a dense wehlike structure was formed. Although crystalline units of polysiloxanes can he observed on the surfaces, the fiber does not seem to he totally covered with the silane even after a very long silanization procedure (reactiontime longer than 15h). Similar results were obtained when deposing GPTS on the chalcogenide fiber surface. Up to now, only morphological surface characterization of silanes has been performed, since the substrates were either (19) Wang, J. J. Electroonal. 1990, 255, 3.
Distinct spectral changes were observed in situ as a consequence of the various immobilization procedures. In general the spectra reported in this section fully agreed with reference spectra presented in other papers dealing with this chemistry on other suhstrates.21.22 The IR-ATR spectra gave valuable information on a molecular basis after each in-situ chemical process, hut for principal reasons (lack of spatial distribution) it wasnotpossihletodetermine the homogeneity of the overall lavers hound to the surface with this method. Therefore othe; surface investigation tools were used to analyze a small area along the derivatized fiber surface. The transmissionspectraofthe mcdifiedfiher surfacetaken with the FTIR microscope present the same features as the ATRspectra takenwith the FTIRspectrometer. In addition, this technique leads to valuable information about the fiber coverage afer each chemical derivatization procedure. The spectra collected were made in a microscopic area of -90-pm diameter. Scanning along the fiber axis showed peak height variations of the silane below 5%, assuring a low lateral roughness in the micrometer range. Hence derivatization under different experimental conditions was tested on fiber samples and spectra were recorded to see whether or not the coating was appropriate and homogeneous. From the secondary electron images and from the EDXray spectra of the silanes, one can see that, on a microscopic scale, silanes present totally irregular microstructures scattered along the chalcogenide surface. The formation of polysiloxane is expected after silanization for a few hours; however, in order to cover the fiber totally, it is important to allow for longer reaction times. A compromisehas to be found between the density of the silane on the fiber surface and the (20) Rugar, D.; Hansms, P. Phys. Today 1990, (Oct),23. (21) Susi, H.; Timasheff, S. N.; Steven, L. J. Bid. Chem. 1967.24, 5460. (22) Weigel. C.; Kellner,R. In ChemicallyModified Surfaces;Leyden,
D. E., Collins, W.T.,E%.; Yark, 1990; p 39.
Gordon and Breach Science Publishers: New
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ANALYTICAL CHEMISTRY, VOL. 65. NO. 17. SEPTEMBER 1. 1993 count.
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images revealed the presence of smooth areas on top of the suhstrates and areas enriched withcompact clusterscomposed of the GOx hound to the surface via 3-APTS and cross-linked with glutaraldehyde. The overall image showed an island structure coverage. Images of GOx entrapped within a polypyrrole coating on a glassy carbon surface were characterized with STM by Yaniv et al.,23 also shown were isolated enzyme clusters, reflecting the heterogeneous microdistrihution of the derivatized layer.
CONCLUSION
-l W A (a. top; b. bottom) AFM image of a chalcogenide Rber darlvatlzed wim 3-APTS/GA/GOx. Figure 8.
relative thickness distribution. Consequently,silanes do not properly cover chalcogenide fibers as is known for other substrate depositions.3 The silicone distribution on the surfacewas not improved even by optimizingthe experimental silanizationconditions. The first topographic AF'M analysis of derivatized chalcogenide fibers was made and the images obtained attested to the feasibility of this investigation at high resolution. Images of high quality were obtained from different scanned areas (approximately 1-4 pm2). The AFM
In general the morphology of the reactive film showed irregularly shaped polymer structures, attesting that aggregates were formed on the surface. The silanes, due to the long reaction time, were cross-linked, resulting in the formationof irregularmultilayers. Theconvolution ofthelayers was not controllable by the reaction conditions since the surface of the fiber is obviously not equally reactive. This paper demonstrates how to characterize deposited layers on chalcogenide glass fibers using complementary surface analysis techniques. Each analysis technique presented here has ita own strengths and limitations; therefore, complementary surface analysis tools are necessary to he able to understand complex chemical modification mechanisms on fairly new materials, for which elucidation of the surface chemistrv is still in an early stage. FTIR spectroscopy delivered information on a molecular basis, whereas SEM and AFM displayed microscopic and nanoscopic topographies of the different derivatization methods. These complementary techniques strongly contributed to the understanding of the phenomena occurring on the waveguide surfaces used for chemical sensors. An extension of the AFM technique to image in different media (e.g., under liquid) will allow new opportunities for in-situ study of chemical surface reactions to monitor the layer-by-layer growth during the construction of a chemical sensor. The surface characterization of chemically modified chalcogenide mid-IR fibers has shown that there is a limitation to using silanes as enzyme carriers to huild a strongly active layer on the fiber surfaceand thatan important improvement ~~
(23) Yaniv, D.;MeCormick, L.;Wang, J.; Naaer. Chem. InterfacialElectroehem. 1991, 353,314.
N.J . Electrooml.
ANALYTICAL CHEMISTRY, VOL. 85, NO. 17, SEPTEMBER 1, 1993
has to be made to maintain a high efficiency of the sensor response. The use of S-layers, bacterial crystalline proteins, for the immobilizationof enzyme on the fiber substrate could lead to a reactive layer with high-packing density of enzyme in one homogeneous monolayer, as has been shown by Sleytr et al.u*B This investigation will be the topic of our next paper.
Acknowledgment is made to the "Fonds zur Forderung der wissenschaftlichenForschung in 6sterreich" for support (24) Pun, D.; Sbra, M.; Sleytr, U. B. J. Vac. Sci. Technol. 1989, B7. (25) Sleytr, U. B.;Meaner, P. Electron Microscopy of Subcellular Dynumics; Plattner, H., Ed.;CRC Press: Boca Raton, FL, 1989.
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of this work (Project 7898). The authors thank Dr. A. Hagenhoff from the Physikalisches Institut der Universitlit Miinster, Germany,for her valuable contribution to the TOFSIMS measurements, Dr. H. F u c b from BASF AG Polymer Research Laboratory, Ludwigshafen, Germany, for his contribution to the AFM measurements, and Dr. K. Miethe, Telekom,Darmstadt, Germany,for making available the PhiAtomica SIMS instrument.
RECEIVED for review March 3, 1993. Accepted May 14, 1993.
