Patterns of functional proteins formed by local electrochemical desorption of self-assembled...

Electrochimica Acta 47 (2001) 275–281

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Patterns of functional proteins formed by local electrochemicaldesorption of self-assembled monolayers

Thomas Wilhelm, Gunther Wittstock *Wilhelm Ostwald Institute of Physical and Theoretical Chemistry, Uni�ersity of Leipzig, Linnestraße 2, D-04103 Leipzig, Germany

Received 5 October 2000; received in revised form 14 March 2001

Dedicated to Professor Dr. Rudiger Szargan (Leipzig) on the occasion of his 60th birthday

Abstract

Patterned self-assembled monolayers (SAMs) were formed using the scanning electrochemical microscope (SECM). Theprocedures is based on the local electrochemical desorption of an alkanethiolate monolayer by applying a 5 kHz square-wavevoltage of 2 V (peak-to-peak) to a two-electrode configuration consisting of an ultramicroelectrode (UME) of 10 �m diameterplaced about 5 �m above a macroscopic SAM-covered gold electrode. Desorption occurs on well-defined regions with a diameterof (12.8�2.8) �m. These regions of bare gold are able to chemisorb a �-functionalized thiol or disulfide such as cystamine to formpatterns of amino-terminated surfaces. Functional proteins can be coupled to the amino groups present at the modified regionsof the monolayer. This approach was demonstrated by imaging the activity of horseradish peroxidase bound to the patternedSAMs in the generation–collection mode of the SECM. A considerable improvement of the procedure could be achieved byperforming the desorption in a solution containing a millimolar concentration of the �-functionalized thiol/disulfide ensuringeffective refilling of the monolayer by the desired molecules and hence high concentration of the immobilized proteins. Themethod is discussed with respect to prospective application in the field of chip-based bioanalytical assays. © 2001 Elsevier ScienceLtd. All rights reserved.

Keywords: SECM; Surface modification; Patterned self-assembled monolayers; Protein arrays; Horseradish peroxidase

1. Introduction

Generation of micropatterns of biomolecules has be-come a very active field of research due to demands forparallel testing systems and continuing development ofminiaturized biosensors and biosensor arrays. WhileDNA-based testing systems have already reached thecommercial market, equally advanced testing systemsusing proteins as functional units are more difficult toconstruct because the proteins typically are much lessstable and may denature upon contact with the surfaceonto which they are to be immobilized. RecentlyMacBeath and Schreiber [1] created arrays of func-tional proteins by transferring drops of an enzymesolution onto an activated glass surface by an arrayerrobot. The proteins were functional with a spot size of

about 150–200 �m in diameter, which was limited bythe surface area wetted by a nanoliter droplet trans-ferred by the arrayer robot. In order to achieve smallerfeatures, several strategies are pursued. They includelocal electropolymerization of conducting polymerswith entrapment of the protein, either on microstruc-tured electrode surfaces [2] or in a microscopic thinlayer configuration using the scanning electrochemicalmicroscope (SECM) [3,4]. Other approaches are basedon local photochemical activation of surface functional-ities [5], on electrocoagulation on microstructured elec-trodes [6], assembling and manipulation of magneticmicrobeads [7–9] or on the use of piezoelectric fluidpumps (ink jet principle) [10].

Patterned self-assembled monolayers (SAMs) havereceived particular attention as a starting point forbuilding biochemically active surfaces. They allow thebinding of functional units in a defined distance to theelectrode which may be used to facilitate direct electron

* Corresponding author. Fax: +49-341-973-6399.E-mail address: [email protected] (G. Wittstock).

0013-4686/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.PII: S 0 0 1 3 -4686 (01 )00566 -7

T. Wilhelm, G. Wittstock / Electrochimica Acta 47 (2001) 275–281276

transfer [11,12] or arrangement of supramolecular cata-lytically active entities on surfaces [13]. Microstructur-ing of such layers has been achieved by the removal ofadsorbates by scanning probe tips [14,15] and by usingan AFM tip as a microscopic pen [16]. The methodswere used to create rectangles of 35 nm2 up to 50×50nm2 [14,15], points with 500 nm diameter [16] or lines30–100 nm wide and 2000 nm long [16]. After bindingof biomolecules to such patterns, the active regionswould be too small for most biosensing application dueto the insufficient immobilized biochemical activity.Microcontact printing has gained popularity for mi-cropatterning of SAMs [17]. A defined fraction of thesurface is wetted with a n-alkanethiol. Rinsing thesurface with a �-functionalized thiol/disulfide forms abifunctional pattern to which proteins can be coupled.Direct stamping of proteins has been reported as well[18,19]. In both cases, all available anchor groupswould be derivatized in one step preventing the forma-tion of arrays with different functional units. SECM hasbeen used to generate reagents that locally convert amonolayer. Subsequent reaction with enzmyes is re-stricted to the regions modified by the SECM-generatedreagent [20]. In our previous work, the SECM has beenused to locally desorb an alkanethiol monolayer in thedirect mode [21]. Subsequent chemisorption of cys-tamine and covalent coupling of glucose oxidase (GOx)lead to an enzyme pattern whose activity was imaged inthe generation–collection mode of the SECM [22]. Asignificant improvement of this concept was achievedby direct application of a square-wave of 10 kHz to atwo-electrode configuration of the macroscopic goldelectrode and the ultramicroelectrode (UME) [23]. Veryrecently, SECM has also been used to generate micro-scopic regions of gold clusters, which could be modifiedby chemisorption of cystamine and subsequent couplingof GOx [24].

This communication uses a sequence of three stepsto build enzyme micropatterns: (i) local electro-chemical modification of self-assembled alkanethiolmonolayers; (ii) refilling the monolayer with an �-func-tionalized thiol; and (iii) covalent coupling of a func-tional protein to the terminal functions of the modifiedmonolayer. Compared to previous reports [21–23] andbesides reducing the spot size further, this paper investi-gates how close regions of electrochemical desorptioncan be placed to each other without compromisingshape and edge definition. The reproducibility of thedesorption step is tested as well. Furthermore, it isdemonstrated for the first time that the local desorptionand refilling of the monolayer can be carried out in oneprocess step if the electrochemical desorption is per-formed in a solution containing the �-functionalizedthiol.

2. Experimental

Gold substrates were prepared by resistive evapora-tion of gold onto Cr-primed glass. Subsequent immer-sion into a 1 mM solution of n-dodecanethiol (Fluka,Buchs, Switzerland) in 2-propanol (Fluka) for 12 hproduced a SAM that was further modified by localdesorption. Cystamine dihydrochloride (Merck, Darm-stadt, Germany), horseradish peroxidase (HRP, Sigma,Deisenhofen, Germany), ferrocenemethanol (ABCR,Karlsruhe, Germany), poly(oxyethylene) sorbitanmonolaureate (Tween 20, Serva, Heidelberg, Germany),and H2O2 (Merck) were used as received.

Two home-built SECM instruments have been usedand were described previously [23,25]. Glass-enclosedplatinum wires of 10 and 25 �m diameter were used asUME. The ratio, RG, between the radii of the glass andthe active electrode area, rT, ranged between 9 and 15and was achieved by grinding and polishing the elec-trodes in a home-built polishing station built accordingto the recommendation of Kranz et al. [26]. A Pt wireas auxiliary electrode and a saturated calomel electrode(SCE) completed the electrochemical three-electrodesetup for SECM imaging experiments and approachcurves. All electrochemical potentials are given withrespect to the SCE if not stated otherwise. Images weregenerated with the in-house developed software MIRA.A linear background was subtracted from the images inFig. 3. Fig. 5 is shown as recorded.

Localized desorption was performed in air-saturated0.1 M KOH solution. The UME was approached to adistance of 1–2 rT with 1.2 �m s−1 by monitoring thedecrease of the oxygen reduction current according tothe approach curves for negative feedback [27]. At fixedUME position, the electrodes were disconnected fromthe potentiostat. The UME and the alkanethiolate-cov-ered gold electrode were directly connected to a func-tion generator DS345 (Stanford Research Systems,USA) and a square-wave voltage (2 V peak-to-peak, 5kHz) was applied to the two-electrode system. Aftercompletion of the desorption experiment, the electrodeswere disconnected, the solution exchanged and copperwas galvanically deposited (5 mM CuSO4+0.1 MH2SO4, −0.2 V vs. Cu�CuSO4 (0.005 M), 30 s) on theregions of the macroscopic gold electrode from whichthe thiols had been desorbed. The electrode with thedeposited Cu was then immersed in a 1% Na2S solutionfor 30 s to give black copper sulfide that can be easilyinspected and measured with a CCD camera connectedto an optical microscope. Full details are given in Ref.[23]. The scale bar (Part-no. O652, Plano GmbH, Wet-zlar, Germany) shown below the microphotographicpictures has a 100 �m line separation and was takenwith the same magnification as the corresponding opti-cal images.

T. Wilhelm, G. Wittstock / Electrochimica Acta 47 (2001) 275–281 277

For continuous desorption/re-adsorption experi-ments, the desorption solution was 50 mM cystamine+0.1 M KOH. The electrode was moved laterally with0.5 �m s−1 while applying a square-wave of 5 V peak-to-peak and 10 kHz. After the desorption, the elec-trodes were rinsed and incubated 2 h with horseradishperoxidase and glutaraldehyde (2 mg HRP in 100 �lbuffer containing 1.25% (v/v) glutaraldehyde) in orderto bind the enzyme to the regions at which thechemisorbed cystamine provided amino groups for at-tachment of the enzymes. Unbound enzyme was rinsedoff with phosphate buffer containing 0.25% (v/v) poly(-oxyethylene)sorbitan monolaureate.

3. Results and discussion

3.1. Local desorption of alkanethiolate SAMs

In a previous communication, we reported that alka-nethiolate SAMs can be electrochemically removed in alocalized manner by applying an ac voltage (10 kHzsquare-wave, 3 V peak-to-peak) between a macroscopicSAM-covered gold electrode and a Pt UME with aradius rT=12.5 �m diameter placed in a distance d ofabout 12 �m above the gold electrode [23]. The regionfrom which alkanethiols were desorbed had a diameterof about 30 �m, which is 1.2 times the diameter of themicroelectrode used. The desorption of the alkanethiolsis caused by the reductive desorption and oxidativedecomposition of the alkanthiolate monolayer which iswell documented for macroscopic samples [28]. Theinclusion of positive potentials chemically transformsthe alkanethiols thus preventing immediate re-adsorp-tion of the desorbed molecules [29].

The desorption process is self-limiting. The desorp-tion starts directly beneath the UME. Once a small areaof the bare gold is formed, additional processes likeoxidation/reduction of the gold itself, solvent electroly-sis and oxygen reduction are contributing to the overallcurrent. In addition the double layer capacity is muchhigher at the bare gold electrode than at the surround-ing SAM-covered gold leading to locally enhancedcharging currents. All these currents have to balancedby the faradaic and charging current at the UME.Because of its limited size, the faradaic currents and thecharging currents at the UME will limit the size up towhich the desorption region can expand at the macro-scopic gold electrode [23]. Considering this mechanism,an open question was how the presence of one desorbedregion influences the local desorption at a new spot inthe immediate vicinity, or, in other words, how closedesorption regions can be placed. For the investigationof this phenomenon, a series of desorption regions wasformed on a SAM-covered macroscopic gold electrode(Fig. 1a). Throughout the experiments the same UME(rT=5 �m, RG=10, d=5 �m) was used with anidentical potential program (38 s, 2 V peak-to-peak, 5kHz square-wave). There are four groups of desorptionspots. From left to right the spot spacing (midpoint-to-midpoint) was varied from 25 (two spots), 20 (twospots), 15 (three spots), 20 �m (three spots). It isobvious that a series with very small spot separationcan be created without compromising shape and edgedefinition of the individual spots. This is also seen in amagnified image of the group with a spot separation of15 �m (Fig. 1b). There is a slight variation of size andshape within the set in Fig. 1b. The diameters of thedesorption spots within the series in Fig. 1a are (12.8�2.7) �m. They were obtained with an UME of 10 �mdiameter. Considering our previous results with a UME

Fig. 1. Optical microscopy of desorbed regions generated in a two-electrode thin layer configuration (d=5 �m) with a microelectrode(rT=5 �m) and a dodecylthiolate-covered macroscopic gold elec-trode. The midpoint-to-midpoints spacings in the four groups arefrom left to right: 25, 20, 15, and 20 �m. The desorbed regions weremade visible by galvanic deposition of copper followed by conversionto black CuS. The scale bare is a microphotograph of an optical scalewith 100 �m line-to-line separation obtained with the same micro-scope setting. (a) Survey of four sets and (b) enlargement of the spotswith a spacing of 15 �m (set is second from right in (a)).


Fig. 2. Detection of immobilized horseradish peroxidase (HRP) activ-ity immobilized on patterned SAM. The sketch is out of scale (seecaption of Fig. 4).

defects of the monolayer. Due to the effective masstransport to such well-separated point defects duringgalvanic copper deposition, the size of the Cu deposit islikely to exceed the actual size of such defects. Thedensity of such defects varies between different batchesof SAM-covered gold substrates despite continuousefforts to enhance the reproducibility of their prepara-tion. The experiments in Fig. 1 also demonstrate that acertain level of defects can be tolerated in this pattern-ing protocol.

3.2. Refilling and functionalization of desorption spots

In an attempt to create a 2×2 spot array modifiedwith the enzyme HRP, local desorption was performedwith 50 �m center-to-center distance between the spotposition. After desorption, the solution was exchangedagainst a 50 mM solution of cystaminium dihydrochlo-ride. After chemisorption (30 min), the solution wasexchanged against a mixture of HRP and glutaralde-hyde to bind the enzyme to the aminoterminated sur-face (120 min). The local enzyme activity wasdetermined in the SECM generation–collection modeas shown in Fig. 2. At the sample, the enzyme-catalyzedreduction of H2O2 proceeds using hydroxymethylfer-rocene (Fc) as electron donor:

at the sample:H2O2+2Fc ��[HRP]

2 OH−+2 Fc+ (1)

At the UME the ferrocinium derivative (Fc+) is de-tected amperometrically:

at the UME: 2 Fc++2 e− ��+50 mV

2 Fc (2)

Fig. 3 shows two image frames recorded with identi-cal setting. Fig. 3c was recorded 6.5 h after Fig. 3b. Thearray is only faintly visible with a slow decrease inactivity during the prolonged recording. A recordingwith no H2O2 added gives a background current of−11.7 to −13.7 pA over the entire image frame (notshown). It is also obvious that the spots in Fig. 3 donot have the same intensity. The first spot was gener-ated in the top-left corner of the array. The one in thebottom-right corner was created last and has thehighest enzyme activity of all four spots. This effect hasbeen observed by us with varying intensity in similarexperiments. It is plausible that desorbed alkanethiols,decomposition products and other impurities in thesolution re-adsorb on the desorption spots before thesample is exposed to the cystamine solution. Therefore,cystamine reacts with surfaces partially blocked alreadyshould be rearranged to surfaces already blocked par-tially by other organic materials. The spot generatedlast contains less adsorbed organics and will chemisorbmore cystamine than the spots generated before. Conse-quently, more amino groups are available for covalentcoupling of the enzyme.

of 25 �m diameter that created desorbed regions ofabout 30 �m diameter, it is likely that the size of thedesorbed regions is proportional to the size of theUME although a slight variation with distance is to beexpected. This effect is difficult to quantitize as we alsofound a rather strong dependence on the initial qualityof the SAM. There is no systematic increase in the sizeof the desorption spots for repeated use of one UME.The spots in Fig. 1b were obtained in the sequencefrom right to left (large to small!). The apparent de-crease in spot size within the series is most likely due toa slight tilt of the sample, from which slightly differentUME-sample spacing result at each position. The num-ber of spots that can be formed with one electrode islimited to about 50 (vide infra). Electrodes used longerfail to produce desorption spots.

The approach shown here differs from the electro-chemical micromachining introduced by Schuster et al.[30]. Schuster et al. [30] achieved the confinement ofelectrochemical reactions by voltage pulses that areshorter than the time constant for charging the electro-chemical double layer (double layer capacitance timessolution resistance). If pulses in the nanosecond timescale are delivered to the two-electrode setup, signifi-cant charging of the double layer (and hence electro-chemical etching or deposition reactions) occurs only atnanometer to micrometer separations of the tool andworkpiece electrodes.

Our procedure works with pulses in the time scale ofabout 200 �s and therefore does not depend on theinstrumentally demanding delivery of the ns voltagepulses to the electrode setup used in [30]. In our casethe localization of the surface modification dependscritically on the decrease of double layer capacitanceand the passivation of the macroscopic electrode by thealkanethiolate SAM. If SAMs show poor inhibition ofmediator recycling in approach curves with the SECMfeedback mode, they do not give good results in local-ized desorption experiments. This is probably due tothe disabling of the local confinement mechanism ex-plained above. The small black dots uniformly scat-tered around the entire image in Fig. 1 represent initial


Fig. 3. SECM image frames from a 2×2 spot array modified withHRP. (a) In scale sketch of the layout. 6.5 h passed between therecording of (b) and (c). The grayscale image was obtained bysubtracting a linear background current from the image to make thesmall current variations visible. Image frame 150×150 �m2, rT=5�m, �T=10.3 �m s−1.

to the terminal groups, it is of great interest to decreasethe number of processing steps for this patterning pro-cedure and to suppress the re-adsorption of alkanethi-ols and other unwanted organic impurities from theworking solution. This was achieved by performingdesorption in a solution that contains a rather highconcentration of �-functionalized thiol/disulfide. Whileapplying an ac voltage between the UME and themacroscopic gold electrode, the microelectrode wasmoved laterally in a constant height above the SAM-modified gold electrode (Fig. 4). Cystamine is immedi-ately available to chemisorb on the bare gold surfaceleft behind the moving UME. Thus a line of an amino-terminated SAM within a methyl-terminated SAM isformed continuously in one process step. By controllingthe movement of the UME, patterns are formed in avery flexible way. After solution exchange HRP wascovalently attached to the amino groups. The activityof HRP was tested with lateral resolution as discussedbefore using the SECM generation–collection mode(Fig. 2). If both H2O2 and hydroxymethylferrocene arepresent in the solution bulk, the local generation of theferrocinium derivatives can clearly be seen from thelarger reduction currents at 50�x�100 �m in Fig. 5a.For the control experiment, the solution was exchangedby removing the working solution and introducingH2O2-free 2 mM hydroxymethylferocene solution (Fig.5b). An image of the identical region shows muchsmaller reduction currents than in Fig. 5a. Please notethat the currents axis extends over 250 pA in Fig. 5aand b. The slightly enhanced reduction currents atx=70 �m in Fig. 5b result from the enzymatic conver-sion of H2O2 that remained in the cell because ofincomplete solution exchange. If the relative position ofUME and sample are to be maintained during solutionexchange, complete solution exchange is not straight-forward in our setup as some liquid is always trappedin inaccessible parts of the cell. The comparison of Fig.5a and b proves the dependence of the signal on theavailability of H2O2 and thus the role of the locallyimmobilized HRP activity in the image formation pro-cess. In conclusion, well-defined microscopic regions ofimmobilized functional proteins can be generated bycontinuous desorption and refilling of a SAM followedby covalent coupling of the biomolecules.

One point of concern with our strategy is the etchingof the used UME. After prolonged desorption experi-ments the quality of the desorption spots decreases. Theoriginal quality is achieved by using a fresh UME. Thepotential program can be optimized in order to achieveeither fast desorption while accepting a short lifetime ofthe UME or in order to prevent the use-up of theUME. In the latter case desorption times per spot of upto 3 min have to be accepted. The potential programused here represents a compromise between the twodemands. It allows to create arrays of up to 50 spots

Fig. 4. Schematic representation of continuous local desorption of analkanethiolate monolayer and refilling by chemisorption of cys-tamine. The sketch is not in scale. In order to illustrate the mecha-nism, the UME-sample distance was enlarged by a factor of 7, thesize of the chemisorbed molecules was enhanced by 4 orders ofmagnitude. No attention was paid to orientation of the alkyl chainsvs. the surface normal and other structural details of the monolayer,which are not of importance for the described experiments.

3.2.1. Continuous desorption and refilling of the SAMAfter demonstrating the possibility to refill desorp-

tion spots with an �-functionalized thiol/disulfide fol-lowed by covalent coupling of functional biomolecules


Fig. 5. Detection of HRP activity in the GC mode. HRP was covalently bound to a line of cystamine generated by continuous desorption andrefilling of the monolayer. (a) In the presence of H2O2, rT=12.5 �m, �T=10.3 �m s−1, 0.5 mM H2O2+2 mM ferrocenemethanol in phosphatebuffer pH=7.0, ET=50 mV; (b) after rinsing out H2O2 and introducing a solution of 2 mM ferrocenemethanol in phosphate buffer.

with one electrode at still acceptable desorption timesper spot.

4. Conclusions

The formation of patterned self-assembled monolay-ers by locally induced electrochemical desorption [21–23] could be considerably improved by performing thedesorption in a solution of the �-functionalized thiol/disulfide which immediately refills the monolayer if theUME is moved to a different location. The availabilityof the locally introduced terminal amino groups in themonolayers and their suitability for binding functionalproteins were demonstrated with the local immobiliza-tion of the enzyme horseradish peroxidase and thesuccessful imaging of its localized activity. By using aUME with 10 �m diameter local spots of (12.8�2.7)�m could be generated. Such procedures open the routefor microscopic modification of surfaces with functionalproteins that are of interest in high-density chip-basedbioanalytical systems. The good shape definition of thedesorption regions should allow to create SAM pat-terns with complex layouts in one process step providedthe microelectrode has the required shape. Recent pro-gress in electrochemical micromachining [30] raiseshopes that such electrochemical tool electrodes can beproduced by electrochemical micromachiningprocedures.

Acknowledgements

The authors are thankful to Professor Dr. R. Szara-gan (University of Leipzig) for his encouraging interestin the progress of this project. The research was fundedby Deutsche Forschungsgemeinschaft (Wi 1617/1-3)and Fonds der Chemischen Industrie (661013).

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