For Imaging of In Situ Enzyme Activity

Angelika Kueng, Christine Kranz, Alois Lugstein, Emmerich Bertagnolli, and Boris Mizaikoff

Summary

For investigation of laterally resolved information on biological activity, techniques for simultaneously obtaining complementary information correlated in time and space are required. In this context, recent developments in scanning probe microscopy are aimed at information on the sample topography and simultaneously on the physical and chemical properties at the nanometer scale. With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy with AFM is demonstrated. This instrumentation enables simultaneous imaging of topography and obtainment of laterally resolved electrochemical information in AFM tapping mode. Hence, topographical and electrochemical information on soft surfaces (e.g., biological species) and polymers can be obtained. The functionality of tip-integrated electrodes is demonstrated by simultaneous electrochemical and topographical studies of an enzyme-modified micropattern.

Key Words: Scanning electrochemical microscopy; atomic force microscopy; enzyme activity; tapping mode; bifunctional scanning probe tips.

1. Introduction

Recent developments in the combination of scanning probe techniques are aimed at complementary, simultaneously obtained information about physical and chemical surface properties with high spatial resolution. Because many biochemically relevant processes are based on redox chemistry and electrochemical conversion of molecules, techniques gathering laterally resolved information on coupled oxidation-reduction processes are of particular interest. The combination of atomic force microscopy (AFM) (1) with scanning

From: Methods in Molecular Biology, vol. 300: Protein Nanotechnology, Protocols, instrumentation, and Applications Edited by: T. Vo-Dinh © Humana Press inc., Totowa, NJ

electrochemical microscopy (SECM) (2-4) is a particularly attractive strategy for achieving complementary electrochemical and topographical information with high lateral resolution in a single time- and space-correlated measurement (5). Microfabrication including focused ion beam (FIB) technology of submicro- and nanoelectrodes integrated into conventional AFM tips is an elegant and versatile approach combining SECM and AFM (6,7). The terms AFM and SECM are used for the instrument as well as the technique. An electroactive area is integrated at an exactly defined distance above the apex of the AFM tip (8), enabling contact mode and tapping mode operation (9). Application of AFM tapping mode (10) is a prerequisite for successfully imaging soft biological samples or polymers (11-13).

To illustrate the functionality and capability of this method, bifunctional probes with integrated electrodes are applied to simultaneously image topographical and electrochemical properties of a biologically active sample in AFM tapping mode. As an example, the activity of the oxidoreductase glucose oxidase immobilized at a periodic micropattern of a soft polymer matrix is electrochemically imaged during mapping of the topography (14). This concept is currently extended to AFM tip-integrated electrochemical biosensors for biomedical applications (15-17).

2. Materials

1. Atomic force microscope equipped with a tapping mode liquid cell (e.g., Nanoscope III; Digital Instruments, Santa Barbara, CA).

2. Bipotentiostat (Model 832A, CH-Instrument, Austin, TX).

3. FIB system.

4. DC-sputterer equipped with a Ti target.

5. RF-sputterer equipped with an Au or Pt target.

6. Vapor deposition polymerization (VDP) system or plasma-enhanced chemical vapor deposition (PECVD).

7. Silicon nitride AFM cantilevers with a length of 200 |im, an integrated pyramidal tip (base: 4 x 4 |im; height: 2.86 |im), and a spring constant of approx 0.06 Nm-1.

8. Micropatterned gold/silicon nitride sample (Quantifoil, Jena, Germany).

9. Chloro-p-xylylene (parylene C).

10. Pt wire (diameter: 1 mm) as counterelectrode.

11. Ag/AgCl reference electrode or oxidized Ag wire (diameter: 1 mm) serving as a quasireference electrode (AgQRef).

12. Insulating varnish (RS Components, Northants UK).

13. Glucose oxidase (150,000 U/g, solid, from Aspergillus niger); store at -20°C.

14. Potassium ferrocyanide.

15. Potassium chloride.

16. 0.1 M Phosphate buffer (pH 7.0); store at 4°C.

17. Glucose.

18. EDP, Elektrodepositionslack Glassophor ZQ 8-43225, Canguard (BASF Farben und Lacke, Münster, Germany).

3. Methods

3.1. Preparation of AFM-SECM Tip

The steps described in Subheadings 3.1.1.-3.1.3. outline the procedure for preparation of the AFM-SECM tip including the metallization and insulation of the cantilever, the FIB cutting process to expose the electroactive area, and the characterization of the tip-integrated electrodes.

3.1.1. Metallization and Insulation of AFM Cantilevers

The silicon nitride AFM cantilevers were DC sputtered (CVC Products) with a 3-nm Ti adhesion layer (working pressure: 6 mtorr; power: 350 W; under argon). Subsequently, a 100-nm uniform gold layer was deposited using an RF-sputterer (working pressure: 6 mtorr; power: 150 W; under argon) to minimize mechanical stress on the cantilevers (see Note 1). To insulate the gold-coated cantilever, 700 nm of parylene C was deposited by VDP (see Note 2) (18). The polymerization process includes the following steps:

1. Initial vaporization at 150°C, 1.0 torr.

2. Cleaving into monomeric radicals by pyrolysis at 680°C, 0.5 torr.

3. Adsorption of the monomeric radical onto the sample (cantilever) surface and simultaneous polymerization.

3.1.2. FIB Milling of AFM-SECM Cantilevers

Figure 1 schematically illustrates the fabrication steps of an integrated frame microelectrode along with the FIB images of the milling processes. All modifications were carried out in a two-lens FIB system (Micrion 2500) utilizing a beam of Ga+ions at 50 keV.

Figure 1A shows the cantilever after coating with the metal layer and the insulation layer. The first FIB-milling step, diametrically opposed cuttings, as illustrated in Fig. 1B, determines the size of the integrated microelectrode. The same cutting procedure was repeated in a second step at the frontal areas of the pyramidal tip after a 90° turn of the cantilever (Fig. 1C). The following milling step (Fig. 1D) comprises reshaping the AFM tip and adjusting its length in correlation with the integrated electrode area. For optimum current responses according to SECM theory (19), the reshaped AFM tip (tip-substrate separation) should be <0.7 x electrode edge length (Fig. 1F). This milling step must be repeated again at the side and the frontal areas of the pillar to reshape the nonconducting original AFM tip. Figure 1E shows the integrated microelec-trode after a final single-pass milling step to remove accumulated material resulting from previous cutting steps from the electroactive surface. Because of the reshaped AFM tip, this procedure ensures high resolution in topography and a precisely defined and constant distance between the integrated electrode

(A) A Si,N4'Cantilever modified with an Au-layer and an insulating layer (Si.N.)

original AFM-canlilcv#r a

Insulation layer J^^ gold layer

{8) Removing both, part of the gold layer arid parts of (he original Si.N.-tip* by diametrically opposed cuttings.

(C) Repeating trie diametrically opposed cuttings at the front areas of the pyramidal Up, after a 90" lum of the cantilever.

{0) Re-shaping the pillar and adjusting the length of the pillar, according to trie size of Ihe electroactive area.

(E) Final milting step in order to remove deposited material.

aultf olectrode radius Inner electrode radius inlegralod framo mferwlectrode insulation lay*r

AFM Up aultf olectrode radius Inner electrode radius inlegralod framo mferwlectrode insulation lay*r

AFM Up

{F) Schematic of Integrated frame electrode with an edge length of 1.5 pm.

Fig. 1. (A-F) Modification steps of coated AFM tip using FIB technique showing (left) schematic view of processing step and (right) corresponding FIB images. (From ref. 6 with permission.)

Fig. 2. (A) FIB images of integrated frame submicroelectrode with edge length of 1500 nm and tip height of 800 nm; (B) cyclic voltammogram recorded at this frame submicroelectrode for oxidation of 0.01 mol/L of K4[Fe(CN)6] in 0.5 mol/L of KCl (scan rate: 100 mV/s).

and the sample surface within the working distance for electrochemical imaging of the surface properties.

3.1.3. Electrochemical Characterization of Tip-Integrated Frame Microelectrodes

Electrochemical characterization of the integrated microelectrodes was done by cyclic voltammetry using 0.01 mol/L of K4[Fe(CN)6] in 0.5 mol/L of KCl as a supporting electrolyte (Fig. 2B). Currently, no theoretical description for square-frame electrode geometries is available in the literature. Development of a suitable theory is in progress. Hence, the theoretical description of ring microelectrodes was used for characterization of the electrochemical behavior. The steady-state current response of a ring microelectrode can be described using Eqs. 1-3 (20,21):

for all other ratios alb. In Eqs. 1-3, D and c are the diffusion coefficient and the bulk concentration of the redox mediator, respectively; F is the Faraday constant; n is the number of transferred electrons; a is the inner electrode radius; and b is the outer electrode radius (see Fig. 1F). Based on Eqs. 1 and 2, the theoretical steady-state current for the oxidation of 0.01 mollL of [Fe(CN)6]4-with n = 1 for the cyclic voltammogram of the frame microelectrode shown in Fig. 2A (inner radius a = 650 nm; outer radius b = 750 nm; ratio alb = 0.867) is calculated to be 1.65 nA (diffusion coefficient: 6.7 x 10-6 cm2ls [4]). The experimentally observed steady-state current is 1.4 nA. However, it has to be considered that the diffusion behavior towards a frame electrode differs from the ring geometry owing to additional effects occurring at the edges of the frame geometry.

3.2. Preparation of Sample

The simultaneous imaging of the topographical and electrochemical properties of a soft biological sample with bifunctional AFM-SECM probes is described. As a test sample, the pores of a periodic microstructure were filled with a soft polymer matrix entrapping the enzyme glucose oxidase (14) (see Note 3).

Figure 3 illustrates the fabrication steps of the sample surface along with AFM images. A periodically micropatterned silicon nitride layer (450-nm layer thickness) was deposited onto a gold-coated silicon wafer (see Note 4). The conductive gold layer was used as a working electrode to electrochemi-cally deposit the glucose oxidase-containing polymer films based on pH shift-induced precipitation (Fig. 3A,B) (22). A solution containing glucose oxidase (5 mglmL of water) and Canguard polymer suspension (70 ^LlmL of water) was stored for at least 30 min at 4°C before it was used. A three-electrode setup was used with an oxidized silver wire operating as the AgQRef, a platinum wire as the auxiliary electrode, and the conductive gold layer of the micropattern as the working electrode. For polymer film formation with the enzyme present in solution, a potential-pulse profile (2200 mV for 0.2 s, 800 mV for 1 s, and 0 mV for 5 s vs AgQRef) was applied 12 times, leading to the precipitation of the polymer in the pores entrapping the enzyme. The sample was rinsed with water and phosphate buffer and stored for at least 12 h at 4°C before analysis with AFM-SECM.

3.3. Simultaneous Topographical and Electrochemical Imaging of Enzyme Activity

The experimental setup and the parameters for AFM-SECM imaging of enzyme activity are described in Subheadings 3.3.1.-3.3.4. This includes a description of the instrumentation, the parameters chosen for AFM tapping mode, and the parameters for simultaneous electrochemical imaging.

Fig. 3. Schematic view of sample and corresponding AFM contact mode images (A,B) before and (C,D) after precipitation of enzyme containing polymer film.

3.3.1. Instrumentation: Experimental Setup

The modified tip was mounted into a Nanoscope III, equipped with a tapping mode fluid cell using the liquid inlet and outlet for positioning a Pt wire as the counterelectrode and an oxidized Ag wire as the AgQRef. The electrical contact for the integrated electrode is provided by the gold spring holding the cantilever in the fluid cell. The spring is pressed onto a small exposed area of the gold layer at the end of the cantilever mount. The contact window was opened by mechanically scratching the insulation layer with a needle or tweezers with sharp, pointy ends. After contacting, the cantilever mount was protected by an insulation varnish applied with a fine microbrush.

An overview of the experimental setup is shown in Fig. 4. The electrochemical experiment is controlled with a bipotentiostat, and the electrochemical signal is read into an additional AD channel of the AFM instrument, which is provided by all conventional atomic force microscopes (see Note 5). Thus, the current measured with the integrated electrode can be directly correlated to the topographical information obtained by the reshaped AFM tip.

split photodiode split photodiode cell modified AFM-tip [

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