Micropatterning proteins and synthetic peptides on solid supports: a

Departments of Electronics and Electrical Engineering, Cell Biology, and Biochemistry, Bio-Mac Ltd.,. University of Glasgow, Glasgow G12 8QQ, U.K...
0 downloads 0 Views 3MB Size
Blotechnol. hog. 1992, 8, 155-160

155

Micropatterning Proteins and Synthetic Peptides on Solid Supports: A Novel Application for Microelectronics Fabrication Technology Stephen Britland,*,+ Enrique Perez-Arnaud,t Peter Clark$$$Brian McGinn,ll Patricia Connolly,t and Geoffrey Moored Departmenta of Electronics and Electrical Engineering, Cell Biology, and Biochemistry, Bio-Mac Ltd., University of Glasgow, Glasgow G12 SQQ,U.K. In this paper, we describe a method for immobilizing proteins and synthesizing peptides in micrometer-dimension patterns on solid supports. Microelectronics fabrication technology was adapted and used to lithographically direct the location of immobilization of proteins on appropriately derivatized surfaces. As examples, we micropatterned the protein bovine serum albumin (BSA) and the enzyme horseradish peroxidase (HRP). The catalytic activity of HRP was shown to be retained after being cross-linked to the support. When coupled with solid-phase peptide synthesis, the technique allowed synthetic peptides to be constructed in patterns again having micrometer dimensions. Synthetic polypeptides, polylysine, were constructed in patterns with dimensions that approached the practical limit of resolution for optical lithography a t 1-2 pm. The patterns of immobilized molecules and synthetic peptides were visualized using histochemical methods together with light and fluorescence microscopy. The protein and peptide patterning technique described here is an advance in the field of bioelectronics. In particular, it should now be possible to devise novel methods for interfacing with biological systems and constructing new devices for incorporation into miniaturized biosensors.

Introduction In the past, various techniques have been employed to pattern proteins and peptides that were both adsorbed to, and bound to, solid supports. This was done using selective denaturizationof adsorbed moleculeswith ultraviolet light (Letourneau, 1975; Kuriyama et al., 19851, using a photolithographic masking and "lift-off" process for enzymeentrapped membranes (Nakamoto et al., 1988) and also by depositionof proteins in solutionusing adapted printing equipment (Kimuraet al., 1988). The most precise method has perhaps been a recently reported technique for the light-driven,lithographically-addressablesynthesisof peptides in patterns having dimensions in the order of tens of microns (Fodor et al., 1991). However, the level of precision and patterning resolution afforded by most of these techniques would preclude their use in applications which may require high-resolution protein patterning. We considered that existingtechnologycould be adapted and used to incorporate biological materials such as enzymes, synthetic biomolecules, immunoglobulins, and even cells onto electronically compatible surfaces with micrometer-scaleprecision. By adopting microelectronic fabrication techniques, thereby benefitting from their proven inherent precision and reliability, a method was devised that allowed this to be done. These bioelectronic devices should attract interest for their possible scientific, medical, and industrial applications.

* Address correspondence tothis author at the followingaddress: Department of Neuropathology, Institute of Neurology, University of London, The National Hospital for Neurology and Neurosurgery, Queen Square, London WClN 3BG, U.K. t Department of Electronics and Electrical Engineering. Department of Cell Biology. 5 Present address: Department of Anatomy and Cell Biology, St. Mary's Hospital Medical School, Norfolk Place, London W2 1PG, U.K. 11 Department of Biochemistry. 8756-7938/92/3008-0155$03.00/0

The techniques used to fabricate these devices are described in this paper, and examples are provided of micropatterning that has so far been achieved.

Materials and Methods SurfacePreparation and Silanation. Glass (Chance Propper) and fused silica (Hireus) microscope slides were cut to the required size and shape (routinely 1-2 cm2) using diamond-tipped tools. After this, they were then cleaned by immersion for 10 min in an 81v/v solution of concentrated HzS04 and HzOz at 80 "C, rinsed in copious amounts of RO water (Reverse Osmosis) (Figure lA), and then thoroughly dried. Positive 51400-31photoresist (Shipley Chemicals) was spun onto the surfaces at 4000 rpm for 20 s and then baked at 90 O C for 30 min. The resist was then illuminated by light transmitted through a photolithographic mask containing a relief image of the desired pattern (Figure 1B). The 364-nmlight source was a quartz-halogen bulb which was operated at 220 V and which produced a power density of approximately 3 X 10-2 mW m-2. Exposed resist was removed using Microposit developer (Shipley Chemicals) by 70 s of immersion followed by a rinse in RO water (Figure 1C). Next, the sample was immersed for 5 min in a 2% v/v solution of dimethyltrichlorosilanein chlorobenzene,followed by two rinses in chlorobenzene(Figure1D). The samplewas then immersed in acetone for 15 min, a step analogous to "liftoff" in IC manufacture, and then rinsed again with acetone followed by RO water. This removed unexposed resist and unreacted dimethyltrichlorosilane (Figure 1E). Following this, the sample was immersed in a 1% v/v solution of 3-[ (2-aminoethyl)aminol propyltrimethoxysilane in a solution of 5 % v/v RO water (pH 5.0) in absolute ethanol for 30 s, followed by 2 rinses in ethanol and a final rinse in RO water (Figure 1F). Finally, the sample was baked at 115 "C for 10 min. The completed micropatterned

0 1992 American Chemical Society and American Instkute of Chemical Engineers

156

Biotechnol. Pmg., 1992, Vol. 8, No. 2

?rgx

A.

lutaraldehyde

X

B.

X

X

x

C.

D.

E.

U

U

F.

G.

Figure 1. A schematic representation of the steps required for the patterning and derivatization of the support (see Materials and Methods for details of the steps). Closed circles denote methylsilane, and open triangles denote aminosilane.

supports contained regions derivatized with methyl and amino functional groups (Figure 1G). Preparation of Fluorochrome-Conjugated HRP. Rhodamine isothiocyanate (RITC) was coupled to horseradish peroxidase (HRP) in a solution of 0.05 M bicarbonate-buffered NaCl (pH 9.2) at a concentration of 10 mg mL-l by dialysis at 1O:l v/v against 100 pg of fluorochrome mL-l also in 0.05 M bicarbonate-buffered saline (pH 9.2). Dialysis was continued for 14 h at 4 "C. The reaction was stopped by exchanging the dialysis buffer with 0.02 M PBS (pH 7.0) at 4 "C. Dialysis was then continued for 2-3 h against the second buffer. Fluorochrome-conjugated HRP was then purified on a Sephadex G-25 column in 0.02 M PBS (pH 7.0), thus removing free RITC. Protein Immobilization. The derivatized supports were immersed in a 2.5% v/v solution of glutaraldehyde in 0.1 M phosphate-buffered saline (pH 7.0) for 1h at 18 "C (Figure 2A). Following this, the supports were rinsed in fresh PBS with the surfaces being prevented from becoming dry at any time. Next, they were immersed in a solution of RITC-conjugated HRP at a concentration of 1mg mL-l in 0.1 M PBS for 1h (Figure 2B). The supports were then removed from the protein solution, rinsed in fresh PBS, and then immersed in an 8 M urea solution for 2 h to remove adsorbed enzyme (Figure 2C). Silver Staining for ImmobilizedBSA. The method used followed that given by Darbre (Darbre, 1986) with slight modifications, as the protein was in the form of a thin layer on the surface. The slides,with the immobilized BSA, were soaked for 30 min in 10% glutaraldehyde (unbuffered) and then briefly rinsed in distilled water. After this, they were immersed for 10 min in an NH3AgN03 solution, prepared as follows. A total of 1.4 mL of fresh NH40H was added to 21 mL of 0.36 % NaOH, and the mixture was agitated vigorously. To this was added 4 mL of 19.4 % AgN03very slowly,with continuousstirring.

Figure2. A schematic representation of the couplingof proteins to a patterned and derivatized support (see Materials and Methods for details of steps).

After the staining period, the NH3-AgN03 solution was removed and replaced immediately with the developer (0.005 % citric acid, 0.019 % formaldehyde in distilled water). (N.B., The discarded NH3-AgN03 solution was treated with a few milliliters of 5 M HC1 to destroy potentially explosive compounds.) The sample was incubated in developer until the pattern was visible. The stained sample was then fixed with 25 % Amf'ii. The fixing step was not very effective, and the pattern started to fade within a few minutes, so the pattern was photographed as soon as possible. When a slide patterned with 3-[(2-aminoethyl)amino]propyltrimethoxysilanealonewas similarly processed, no evidence of staining was observed. Demonstrating Retention of Catalytic Activity of Patterned HRP. The diaminobenzidine reaction (DAB) for peroxidase was used to demonstrate retention of enzyme activity after immobilization. DAB was dissolved at 0.5 mg mL-l in 0.05 M Tris-HC1 buffer (pH 7.6). Following the addition of H202 at 1%v/v in the DAB solution, the supports were immersed in the resultant solution for 5 min. This brief period was sufficient for an insoluble dark-colored (opaque) reaction product to be formed at the location of the immobilized HRP. Image intensification was completed by precipitation of nickel/ cobalt salts from the indicator solution. DAB kits for the purpose of HRP localization are available commercially. Peptide Synthesis and Fluorochrome Labeling. The peptides were synthesized in a manner which conformed broadly with the principles of Merrifield peptide synthesis (Merrifield, 1963). A schematic representation of the synthesis protocol is shown in Figure 3. The silanated surface was treated with 20% piperidine in DMF (NJV-dimethylformamide)for 2 min followed by washing with DMF. (Fmoc)Lys(Fmoc)-OH[(9-fluorenylmethoxycarbonyl)lysine, 0.3 mmol, 177 mg] and HOBT (l-hydroxybenzotriazole, 0.6 mmol, 81 mg, 2 equiv) were dissolved in DMF (2 mL). This solution was cooled to 0 "C; DIC (Nfl-diisopropylcarbodiimide, 0.33 mmol, 42 mg, 1.1equiv) was added, and the mixture was allowed to warm to room temperature over 15min with stirring. The

157

Biotechnol. hog., 1992, Vol. 8, No. 2 F - moc I F-moc-Lys-OH

+

HOBT

1

DIC

I

F - moc - Lys - OBT (ester)

I

~ N M L H

/

F - moc

/

NH

c=o \

/ CH

NH2

/

# NH2

NH2

H-Lys,

1

Figure 4. The chemical structure of a single synthetic polylysine peptide. PIPERIDINE

NH2

Figure 3. The synthesis protocol for building a polylysine peptide.

silanated surface was treated with the resultant active solution for 30 min and then washed in DMF. The surface was then treated with 20 % piperidine in DMF for 10 min to remove Fmoc groups and was washed again in DMF. The treatments with active ester solution and piperidine solution were repeated an additional two times, and the surfaceswere finally washed with methanol and dried. An expanded diagram representing the intended chemical structure of an individual synthetic polylysine peptide prior to fluorochrome labelingand the position of the peptides on a silane-patterned surface are shown in Figures 4 and 5. Terminal amino groups on the lysine "tree" were labeled with fluorescent molecules to permit visualization by microscopy. To do this, the surfaces were treated with a solution of NaC03 buffer (pH 9.0) containing RITC at a concentrationof 0.1 mg mL-l for 12h a t 4 "C. The surfaces were rinsed copiously in DMF to remove unbound rhodamine. Cell Culture. The cell type used in this study was the fibroblast-like baby hamster kidney cell (BHK-21), a colony of which was maintained routinely in our lab. The basic culture medium was the Glasgow modification of Eagle's minimal essential medium supplemented with 3 mM glutamate, 100 units mL-1 penicillin, 100 pg mL-l streptomycin, 2.5 pg mL-l ampherotericin-B, 10% calf serum, 10% tryptose phosphate broth, and 20 mM Hepes saline. Cell suspensionswere obtained by detachment of the cell from tissue culture plastic universals with 0.05 % trypsin in 0.2 mg mL-l EDTA after rinsing in Ca2+-Mg2+free 20 mM Hepes-buffered Hank's basic salt solution. Trysinization was stopped by the addition of serumcontaining medium. The cells were then washed by centrifugation, resuspended in medium, triturated, and finally counted. The cells were plated from suspension at a density of 1X 105cm-2onto the patterned surfaces and placed in an incubator for 1 h at 37 "C. The surfaces were then immersed twice in basic salt solution as a standard rinse to remove unattached cells. Following this, the patterned substratum was reimmersed in culture medium and returned to the incubator where it was maintained at 37 "C for 24 h. The preparation was then removed from the

Figure 5. A diagrammatic representation of the structure of the peptide shown in Figure 4 and the intended location of peptides on a patterned support.

incubator and photographed using an inverted phasecontrast light microscope. Fluorescence Microscopy. All fluorescence micrographs were taken using a lOOX oil-immersion,high numericalaperture objective on Vickers epifluorescence light microscope. Standard UV illuminationwas used together with a 455-490-nm pass band excitation and a 500-nm + longwave pass band emission filter set.

Results Preparation of the supports to the stage at which protein immobilization and peptide synthesis could commence (Figure 1G) took approximately two hours. An additional two-three hours were required to immobilize the proteins and remove adsorbed materials by a urea soak. A micrograph of light transmitted through a lithographic mask containingthe gratingpattern that was used to create the protein and peptide patterns is shown in Figure 6A. The narrowest bright strips are approximately 1.5 pm in width. Figure 6B shows silver-stainedBSA (dark-colored areas) patterned using the lithographic mask illustrated in Figure 6A. The BSA was patterned at a resolution approaching the practical limit for conventional lithography at 1.5 pm (narrowest strip). The light strips represent areas derivatized with methylsilane to which the BSA was not bound. The fluorescence micrographs in Figure 6C,D show that the HRP was patterned similarly to BSA using both bright-field (C) and dark-field (D) lithographic masks. It was of prime importance to establish that the 8 M urea solution used to remove ad-

158

Biotechnol. Reg., 1992, Vol. 8, No. 2

Figure 7. (A) A support containing patterned HRP before the application of DAB indicator substrate. (B) The same surface 5 min after the application of the DAB substrate. The dark strips define areas of accumulation of reaction product formed through the catalytic activity of the HRP. This confirmed that the enzyme remained active after the immobilizationprocedure and the removal of adsorbed enzyme by a urea soak. Scale bar = 10 pm.

Figure 6. (A) A micrograph showing light transmitted through the lithographic mask comprisinga grating pattern that was used in the preparationof the supports. (B)A micrographof patterned and silver-stained bovine serum albumin. (C) A bright-field fluorescencemicrograph showing patternedRITC-labeledHRP. (D) A dark-field version of the micrograph shown in panel C. Scale bar = 10 pm.

sorbed protein did not render enzymes, such as HRP, inactive. In Figure 7, two photomicrographs show a support containing micropatterned HRP, both before (A) and after (B) the application of DAB indicator solution. The location of the immobilized HRP was invisible before

the application of the indicator solution (Figure 7A). After 5 min, the action of the immobilized HRP on the DAB indicator was to produce a dark-colored precipitate localizing the HRP to regions of the surface corresponding with aminosilane (Figure 7B). Thus, the catalytic activity of the enzyme had therefore been retained. Synthesis of the polylysine peptides took around 3 h to complete. Figure 8 is a fluorescence micrograph showing bright strips containing RITC-labeled synthetic peptides. The width of the narrowest bright lines is again around 1.5 pm. The broader dark bands show that background adsorption of lysine and free rhodamine was minimal. We have begun to use the patterned surfaces in studies on factors affecting cell behavior in vitro. Figure 8 illustrates how the surfaces can be used as models for creating a precisely defined microenvironment for cell culture. The artificial features have approximatelycellular dimensions and can be used to study mechanisms of cell behavior. The alignment of BHK cells in Figure 9 shows how they have responded to the surface chemistry of the patterned substratum. The exact nature of the cell-tosubstratum interaction that induces the cell guidance illustrated in Figure 9 is uncertain. It may represent a charge-dependent effect that is mediated through selective absorption of cell adhesion proteins, for example, fibronectin, from the culture medium to areas treated with aminosilane. There is also the possibility that the guidance depends on a cell receptor-substratum ligand interaction. However, this is considered to be less likely.

Discussion We have devised a technique for immobilizing and constructing actual and synthetic biological molecules in

Biotechnol. Rog., 1992, Vol. 8, No. 2

Figure 8. A fluorescence micrograph showing the location of RITC-labeled synthetic polylysine peptides on a patterned surface. Scale bar = 10 pm.

Figure9. (A) A micrograph of light transmitted through agrating pattern photolithographic mask. (B) BHK cells aligned along adhesive tracks of aminosilaneprepared using the grating mask. Scale bar = 40 pm.

a predetermined configuration on electronically compatible planar surfaces. The methodology involves fabrication techniques that are currently used by the semiconductor industry to manufacture microelectronic circuits. The standard photolithographic process was only slightly modified, and both the duration of the process and the reagents and materials necessary are compatible with those that are currently required for conventional semiconductor fabrication. The grating pattern was used because of its illustrative potential; however, it is possible to produce any twodimensional configuration. The resolution of patterning that can be achieved using this lithographic process is dictated by the wavelength of the illuminating light. The current industry standard for UV lithography is approximately 1.5 pm (Navon, 1986). It is conceivable that optical lithography could be substituted with X-ray and electron-

159

beam lithographies (Wilkinson, 1989), thereby allowing patterning resolution to be increased to submicrometer and even nanometer levels. Attempts have already been made to immobilize proteins in nanometer-scale arrays using suitably prepared surfaces (Douglas et al., 1990). However, poor methodological reliability meant that only a small proportion of available molecules were successfully positioned. Another method, positioning molecules with nanometer precision using a scanning tunneling microscope (Eigler and Schweizer, 1990; Utsugi, 1990), has not yet been applied to biological molecules such as proteins. Immobilization of proteins, glycoproteins,and peptides using cross-linkers is a technique that is employed frequently (Aplix and Hughes, 1981; Bhatia et al., 1989; Burteau et al., 1990). However, our approach, that of patterning immobilized molecules, is novel and of potential importance and applicability in many areas. It is based upon a technique for patterning the surface chemistry of silicon (Kleinfeld et al., 1988). We were able to illustrate that proteins were not denatured during immobilization by showingthat micropatterned HRP retained its catalytic activity. We have since found that it is possible to micropattern molecules other than proteins by using alternative cross-linkers. It should therefore be possible to micropattern both proteins and nonbiological molecules on the same support. Recently,there have been theoretical (Tedesco et al., 1989) and descriptive reports (Ho and Reichnitz, 1987)on the possible use of molecular receptors and artificial enzymes in biosensors. We have demonstrated here that artificial molecules can also be constructed in micropatterns, in this case synthetic peptides. A report has recently described the use of photolithography in a light-directed, spatially-addressable, parallel synthesis system for producing high-density arrays of separate chemical compounds (Fodor et al., 1991). Briefly, an aminosilanated support was activated and effectively patterned simultaneously, by the removal of photolabile protecting groups from specific areas of the support using light transmitted through a lithographicmask. Synthetic peptides were then constructed by chemical coupling of amino acids to specific areas of the support that had been patterned previously by the initial photo-deprotection. The resultant support-amino acid solution-lithographic mask “sandwich”caused the spatial addressability of the system, and therefore the minimum array dimension, to be restricted to around 20 pm due to light diffraction. Nevertheless, this system still permitted 4.0X 104synthesis sites per square centimeter. It is interesting to consider that if a similar “photosynthetic” technique were to be performed on a micropatterned methylsilane/aminosilane support, such as the type described in the present report, the limiting effect of light diffraction on the array dimensions could be eliminated. Theoretically, it should then be entirely feasible to reduce the minimum array dimension to around 2 pm2, which represents the practical limit of resolution for optical lithography. This adaptation to the “photosynthetic”method would increase the number of potential synthesis sites by a factor of 10, giving approximately 4 X 106 cm-2. As it stands at present, the current technique is directly applicableto obtaining precise geometric patterns of proteins for use, for example, in the fabrication of single-typeenzyme sensorson semiconductor materials. Much of the proposed market for biosensors currently at the design or development stage is in both in vivo monitoring (Hunter, 1989) and in vitro testing (Clarke et al., 1985). We feel that many such devices based on conductimetricassay (Watsonet al., 1987),optical assay (Seitz,

Bbtechnd. pros., 1992, Vol. 8, No. 2

160

1989; Magill et al., 1990), or immobilized whole cells or microorganisms (Miller et al., 1989; Korpela et al., 1989; Parce et al., 1989) could perhaps exploit this protein patterning technique. In conclusion, we have devised a method for micropatterning proteins and synthetic peptides on solid electronically compatible planar surfaces. We believe that the techniqueitself is new, althoughit encompassesestablished methods. The ability to pattern proteins at this scale should be very useful if applied to problems related to developmental and cell biology; however, it will perhaps be most relevant to the development of miniaturized sensors.

Acknowledgment Financial support from the SERC is gratefully acknowledged. The technical assistance given by Bill Monaghan, Mary Robertson, Lois Hobbs, Stephen Durr, and George Kaims was much appreciated. Literature Cited Aplin, J. D.; Hughes, R. C. Protein Derivatized Glass Coverslips for the Study of Cell-to-Substratum Adhesion. Anal. Biochem. 1981,113,144-148. Bancroft, J. D.; Cook, H. C. Manual ofHistologica1 Techniques; Churchill Livingstone: London, 1984. Bhatia, S. K.; Shriver-Lake, L. C.; Prior, K. J.; Georger, J. H.; Calvert, J. M.; Bredehorst, R.; Ligler, F. S. Use of ThiolTerminal Silanes and Heterobifunctional Cross-Linkers for Immobilization of Antibodies on Silica Surfaces. Anal. Biochem. 1989,178,408-413. Burteau, N.; Burton, S.; Crichton, R. R. Stabilization and Immobilization of Penicillin Amidase. FEBS Lett. 1990,258, 185-189. Clarke, D. J.; Calder, M. R.; Carr, R. J.; Blake-Coleman, B. C.; Moody,S. C.; Collinge, T. A. The Developmentand Application of Biosensing Devices for Bioreactor Monitoring and Control. Biosemors 1985,1, 213-320. Darbre, A.Practica1 Protein Chemistry, A Handbook;John Wiley and Sons: London, 1986. Douglas, K.; Clark, N. A,; Rothschild, K. J. Biomolecular Solid State Nanoheterostructures. Appl. Phys. Lett. 1990,56,692694. Eigler, D. M.; Schweizer, E. K. Positioning Single Atoms with a Scanning Tunneling Microscope. Nature 1990,344,524-525. Fodor, S. P. A.; Leighton Read, J.; Pirrung, M. C.; Stryer, L.; Tsai Lu, A.; Solas, D. Light Directed, Spatially Addressable Parallel Chemical Synthesis. Science 1991,251,767-773. Ho, M. Y.; Rechnitz, G. A. Highly Stable Biosensor Using an Artificial Enzyme. Anal. Chem. 1987,59,563-537. Hunter, K. W. Biosensors. A New Analytical Technology for Real-Time, On-Line Biochemical Monitoring. Am. J. Clin. Pathol. 1989,91(4),S32-33.

Kimura, J.; Kawana, Y.; Kuriyama, T. An Immobilized Enzyme Membrane Fabrication Method Using an Ink-Jet Nozzle. Biosemors 1988,4,41-52. Klebe, R. J. Cytoscribing: A Method for Micropositioning Cells and the Construction of Two- and Three-Dimensional Synthetic Tissues. Exp. Cell Res. 1988,179,362-373. Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. Controlled Outgrowth of Dissociated Neurons on Patterned Substrates. J.Neurosci. 1988,8,4098-4120. Korpela, M.; Mantsala, P.; Lilius, E. M.; Karp, M. Stable LightEmitting Escherichia coli as a Biosensor. J. Biolumin. Chemilumin. 1989,4,551-554. Kuriyama, J.; Kimura, J.; Kawana, Y. A Single Chip Biosensor. NEC Res. Dev. 1986,78,1-5. Letourneau, P. C. Cell-to-Substratum Adhesion and Guidance of Axonal Elongation. Dev. Biol. 1975,44,92-101. Magill, J. V.; Zhou, Y.; Laybourne, P. J. R.; De La Rue, R. M. The Use of Ion-Exchanged Waveguides in Integrated Optical Molecular Biosensors. Proceedings of the 1stWorld Congress on Biosensors, Singapore, 1990. Merrifield, B. The Synthesis of a Tetra-Peptide. J. Am. Chem. SOC. 1963,85,2149-2154. Miller, A. 0.; Menozzi, F. D.; Dubois, D. Microbeads and Anchorage-Dependent Eukaryotic Cells: The Beginning of a New Era in Biotechnology. Adv. Biochem. Eng. Biotechnol. 1989,39,73-95. Nakamoto, S.; Ito, N.; Kuriyama, T.; Kimura, J. A Lift-off Method for Patterning Enzyme-Immobilized Membranes in Multibioeensors. Sem. Actuators 1988,13,165-172. Navon, D. H. Semi-conductor Microelectronics and Materials; Holt, Rinehart and Wilson: New York, 1986. Parce, J. W.;Owicki, J. C.; Kercao, K. M.; Signal, G. B.; Wada, H. G.; Muir, V. C.; Bousse, L. J.; Ross, K. L.; Sikic, B. I.; McConnell, H. M. Detection of Cell AffectingAgents with a Silicon Biosensor. Science 1989,246,243-247. Seitz, W. R. Transducer Mechanisms for Optical Biosensors.Part 1: The Chemistry of Transduction. Comput. Methods Programa Biomed. 1989,30,9-19. Tedesco, J. L.; Krull, U. J.; Thompson, M. Molecular Receptors and Their Potential for Artificial Transduction. Biosemors 1989,4,135-167. Utaugi, Y. Nanometer-Scale Chemical Modification Using a Scanning Tunnelling Microscope. Nature 1990,347,747-749. Watson, L. D.; Maynard, P.; Cullen, D. C.; Sethi, R. S.; Brettle, J.; Lowe, C. R. A Microelectronic Conductimetric Biosensor. Biosemors 1987,3,101-115. Wilkinson, C. D. W. Nanofabrication for Nanoelectronics and Bioelectronics. In Molecular Electronics Science and Technology; Aviram, A,, Ed.; United Engineering Trustees Inc., 1989;pp 129-136. Accepted January 6,1992. Registry No. HRP, 9003-99-0.