Elsevier

Bioelectrochemistry

Volume 60, Issues 1–2, August 2003, Pages 115-121
Bioelectrochemistry

Imaging of enzyme activity by scanning electrochemical microscope equipped with a feedback control for substrate–probe distance

https://doi.org/10.1016/S1567-5394(03)00055-0Get rights and content

Abstract

The enzymatic activity of diaphorase (Dp) immobilized on a solid substrate was characterized using a scanning electrochemical microscope (SECM) with shear force feedback to control the substrate–probe distance. The shear force between the substrate and the probe was monitored with a tuning fork-type quartz crystal and used as the feedback control to set the microelectrode probe close to the substrate surface. The sensitivity and the contrast of the SECM image were improved in the constant distance mode (distance, 50 nm) with the shear force feedback compared to the image in the constant height mode without the feedback. By using this system, the SECM and topographic images of the immobilized diaphorase were simultaneously measured. The microelectrode tip used in this study was ground aslant like a syringe needle in order to obtain the shaper topographic images. This shape was also effective for avoiding the interference during the diffusion of the enzyme substrates.

Introduction

In recent years, the development of analytical methods for biological materials such as enzymes and living cells has received much attention in advanced biotechnology. Among the various requirements for these analytical methods, acquiring the spatial distribution of the protein with a high sensitivity and selectivity is of particular importance for many applications such as enzyme-linked immnosorbent assays (ELISA) [1], [2], [3]. In the past, optical methods based on the fluorescence or chemiluminescence detection have commonly been used for this purpose. However, miniaturization of the optical instruments is difficult because of the complicated optical systems including lens, a photon detector, light source and dark chamber. In contrast, electrochemical probes have advantages of small size and simplicity. In particular, the scanning electrochemical microscope (SECM) has been found to be suitable for trace analyses required for biochips applications [4], [5]. SECM is a kind of scanning probe microscope (SPM), which detects localized electrochemically active species to afford two- or three-dimensional images based on the distribution of these species [6]. However, a major drawback of the SECM is the difficulty in controlling the sample–probe distance, which decisively governs the sensitivity and the quality of the SECM image. SECM measurements have been conventionally done in the constant height mode, in which the microelectrode probe is scanned without a feedback loop to the probe tip. During imaging in the constant height mode, setting the probe tip close to a rough surface is difficult, resulting in low spatial resolution of the images.

Recently, the feedback distance control utilizing shear forces exerted between the probe and the sample has been applied to the scanning near-field optical microscope (SNOM). For the detection of shear force, many researchers have adopted the tuning fork-type quartz crystal resonators which have a relatively simple mechanism compared with an AFM-like system using an optical lever. For the SECM systems, a tuning fork quartz crystal has recently been used for feedback control of a microelectrode probe to afford the constant-distance imaging [7], [8], [9], [10].

In this study, we use an SECM equipped with the feedback control system and imaged the enzymatic activities for immobilized diaphorase (Dp) in the constant distance mode. Diaphorase was detected by using ferrocenyl methanol (FMA) as the electron mediator. FMA was oxidized to FMA+ at the microelectrode probe and diffused to the immobilized diaphorase, which catalyzed the oxidation of NADH to regenerate FMA. Since the reaction rate of this “redox cycling” is strongly influenced by the diffusion length of FMA and FMA+, the distance between the microelectrode and the surface of the immobilized diaphorase is a decisive factor governing the sensitivity and quality of the SECM image. Electrochemical methods for the biochips based on the detection of enzymatic activities will be greatly improved by keeping the microelectrode very close to the surface in the constant-distance mode. In addition, since the vertical movement of the probe directly reflects the topography of the sample, the simultaneous measurement of the electrochemical and topographic information of the sample can be made by using the feedback distance control.

Section snippets

Chemicals

Diaphorase (Dp) purified from Bacillus stearothermophilus (EC 1.6.99.-) was purchased from Unichika. This enzyme has a molar mass of about 30,000 and has one flavin mononucleotide (FMN) per molecule as an electroactive site [11]. Ferrocenylmethanol (FMA) was purchased from Aldrich Chemicals and recrystallized from hexane. All other chemicals including the β-nicotinamide-adenine dinucleotide, the reduced form (NADH, Orient Yeast), were used as received. All the solutions were prepared using

Characterization of the SECM probes

Fig. 3a shows the typical dependency of the amplitude of the induced signal on the vibration frequency for the prepared SECM probe. Since the absolute amplitude of the induced signals was different from each probe, the amplitude was expressed by an arbitrary unit. As shown in this figure, the maximum amplitude was observed at the frequency of 30.84 kHz, which would correspond to the mechanical resonance of the tuning fork. Although this frequency showed little scattering for each tuning fork

Conclusions

Diaphorase immobilized on the Au array electrode was imaged with an SECM system equipped with the feedback control of the sample–probe distance by monitoring the shear force exerted between the sample and the probe. The feedback control system allows the microelectrode probe to move closer without hitting the sample surface. The SECM measurement of the immobilized enzyme provided information on the enzymatic activity in the local area of the sample. The current response increased as the

Acknowledgements

This work was supported by a Grant-in-Aid for Priority Area 417 (No. 14050010) and a Grant-in-Aid for the Creation of Innovations through Business–Academic–Public Sector Cooperation (No. 13504) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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