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Fig. 4. (A) Experimental setup and (B) scheme of simultaneous AFM-SECM imaging and involved reactions at surface of micropatterned sample with integrated electrode operating in generation-collection mode.

3.3.2. Tapping Mode Imaging

For simultaneous topographical and electrochemical imaging of the enzyme activity, the integrated electrode is scanned over the sample surface in AFM tapping mode using the DI tapping mode liquid cell filled with air-saturated phosphate buffer. The parameters for AFM tapping mode imaging are as follows:

5. Sampling rate: 256 x 256.

The integral gain and proportional gain should be optimized for every measurement so that the height image shows the sharpest contrast and there are minimal variations in the amplitude image (the error signal).

3.3.3. Electrochemical Imaging

For localized detection of glucose oxidase activity, the generation-collection mode of SECM (23) has been applied. In the presence of the substrate glucose, H2O2 is locally generated as a byproduct of the enzymatic reaction. Glucose oxidase catalyzes the oxidation of glucose to gluconolactone, and the electron acceptor oxygen of the enzymatic reaction is reduced to H2O2 (Fig. 4B). H2O2 is directly detected at the integrated electrode using amperometry at a constant applied potential of 750 mV vs AgQRef. A schematic of the involved reactions and the measurement principle is given in Fig. 4B. The parameters for electrochemical imaging are as follows:

1. Technique: amperometry.

2. Counterelectrode: Pt wire.

3. Quasi-reference electrode: oxidized Ag wire.

4. Tip potential: 750 mV vs AgQRef.

5. Sample interval: 0.0025 s.

6. Liquid: for negative control, phosphate buffer (0.1 M, pH 7.0), air saturated; for positive control, add 50 mM glucose.

3.3.4. Simultaneous AFM-SECM Imaging

The tip was engaged to the surface and the image quality optimized (see Subheading 3.3.1.) showing the topographic features of the polymer spots. Subsequently, the potential was applied to the integrated electrode and the current was recorded as a negative control image (Fig. 5A-C). In the absence of glucose in solution, the current recorded at the electrode during AFM tapping mode imaging of the enzyme containing polymer spots was negligible and

Fig. 5. Simultaneously recorded height and current images of glucose oxidase activity in AFM tapping mode with free-drive amplitude of 5 V and drive frequency of 32.4 kHz. Top view of the height (A,D), corresponding amplitude (B,E), and simultaneously recorded current (C,F) images recorded in air-saturated phosphate buffer (0.1 mol/L, pH 7.4) in the absence (A-C) and presence (D-F) (50 mM) of the substrate glucose in solution is shown. The tip was held at a potential of 750 mV vs AgQRef. Electrode edge length: 770 nm; tip height: 700 nm. (From ref. 14 with permission.)

Fig. 5. Simultaneously recorded height and current images of glucose oxidase activity in AFM tapping mode with free-drive amplitude of 5 V and drive frequency of 32.4 kHz. Top view of the height (A,D), corresponding amplitude (B,E), and simultaneously recorded current (C,F) images recorded in air-saturated phosphate buffer (0.1 mol/L, pH 7.4) in the absence (A-C) and presence (D-F) (50 mM) of the substrate glucose in solution is shown. The tip was held at a potential of 750 mV vs AgQRef. Electrode edge length: 770 nm; tip height: 700 nm. (From ref. 14 with permission.)

revealed no electrochemical features (Fig. 5C). For mapping the enzyme activity, the tip was retracted and the fluid cell was filled with phosphate buffer containing 50 mM glucose. After reengagement of the tip, the scanning procedure was repeated. In the presence of glucose, an increase in the current recorded at the nanoelectrode was obtained owing to the localized production of H2O2 when the tip was scanned across the glucose oxidase containing polymer spots (Fig. 5F). The periodicity of the pattern in the electrochemical image corresponds well with the topography provided by the integrated AFM tip. Furthermore, the topography features in the negative and positive control are unaltered but do not give any information about the immobilized enzyme activity.

center frequency - 32 kHz, 1kHz/div

Fig. 6. Frequency spectrum of modified cantilever coated with 100 nm of Au and insulated with 700 nm of parylene C.

center frequency - 32 kHz, 1kHz/div

Fig. 6. Frequency spectrum of modified cantilever coated with 100 nm of Au and insulated with 700 nm of parylene C.

4. Notes

1. Alternatively, a 50-nm Pt layer can be deposited as an electrode material (working pressure: 6 mtorr; power: 30 W; under argon). Higher power results in bending of the cantilever.

2. The metallized cantilevers can also be insulated by applying an 800-nm-thick layer of silicon nitride deposited by PECVD. Dense and well-defined insulation layers are particularly achievable when the process is repeated several times (6 x 5 min PECVD deposition, resulting in approx 800 nm of Si3N4) (6).

3. The described immobilization protocol can be applied to a number of other proteins. For example, entrapment of catalase, lactate oxidase, pyrovate oxidase, NAD+-dependent glucose dehydrogenase, pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase, quinohemoprotein alcohol dehydrogenase, and alcohol dehydrogenase has been reported in the literature (22).

4. The periodically micropatterned wafer was purchased from Quantifoil. Any micro-electrode array can be used for this purpose.

5. Digital Instruments offers a phase box for reading in up to four additional signals.

6. Despite deposition of 100 nm of gold and 700 nm of parylene C, the properties of the initial silicon nitride cantilever remain virtually unaltered compared with unmodified Si3N4 cantilevers (9). Anyway, the optimal drive frequency varies for different samples, fluids, fluid volumes, and cantilevers, and, hence, the drive frequency has to be chosen manually following the DI operation instructions. The best frequencies appear to be on the side of a peak or in a shallow valley between peaks. A typical frequency spectrum of a modified cantilever is depicted in Fig. 6. The regions circled show typical operation frequencies that produce good fluid tapping images.

7. The drive amplitude is adjusted until the desired cantilever response amplitude is obtained. For samples with small variations in height, usually drive amplitudes of 2 to 5 V are required. Good results are usually not obtained on soft samples if it is necessary to use drive amplitudes above 10 V.

8. In general, low scan rates of 1 Hz or lower are advantageous in order to avoid convective effects.

9. The set point can be adjusted by monitoring the image quality. Increase the set point in small increments until the cantilever pulls off the surface. Then reduce the set point in small increments until an image appears and the image is optimized. Usually the best images are obtained at set points just below where an image appears.

Acknowledgments

This work was supported by the National Science Foundation (grant 0216368 within the program Biocomplexity in the Environment), the National Institute of Health (grant EB00058), and the Fonds zur Förderung der wissenschaftlichen Forschung Austria (grants P14122-CHE and J2230).

References

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