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A novel ISFET-type biosensor based on P450 monooxygenases.
Masayuki Hara1, Yoshiaki Yasuda, Hideki Toyotama
1National Institute of Bioscience and Human-technology (NIBH), Agency of Industrial Science and Technology (AIST), Ministry of International Trade and Industry (MITI), Tsukuba, Japan. masayuki-hara@aist.go.jp
Biosensors & Bioelectronics
|February 13, 2002
Summary
We developed a novel biosensor using P450 monooxygenase immobilized on an ion-sensitive field-effect transistor (ISFET). This electrochemical biosensor detects enzymatic activity, showing potential for sensing chlorophenol compounds.
Area of Science:
- Biotechnology
- Biosensor Technology
- Enzyme Engineering
Background:
- Ion-sensitive field-effect transistors (ISFETs) are established electrical devices, primarily used as pH sensors.
- P450 monooxygenases are crucial enzymes with high enzymatic activity, often engineered for specific applications.
- Previous biosensing platforms have not utilized P450 monooxygenases immobilized on ISFETs.
Purpose of the Study:
- To create the first electrochemical biosensor utilizing P450 monooxygenases immobilized on an ISFET.
- To demonstrate the biosensor's ability to detect P450 monooxygenase activity through an electrochemical signal.
- To explore the potential application of this biosensor for detecting chlorophenol compounds.
Main Methods:
- Genetically engineered rat CYP1A1 P450 monooxygenase fused with yeast NADPH-cytochrome P450 oxidoreductase was expressed in Saccharomyces cerevisiae.
- Yeast microsomal membranes containing the enzyme were immobilized on an ISFET device using an agarose layer.
- The enzymatic conversion of 7-ethoxycoumarin to 7-hydroxycoumarin was monitored electrochemically via voltage changes and fluorescence.
Main Results:
- The ISFET biosensor exhibited a voltage increase correlated with P450 monooxygenase enzymatic activity.
- The observed voltage increase was inhibited by MnCl2, a known P450 monooxygenase inhibitor, confirming enzyme-specific detection.
- A positive correlation was established between the ISFET's voltage output and fluorescence measurements of 7-hydroxycoumarin formation.
Conclusions:
- This study presents the first successful electrochemical biosensing platform based on P450 monooxygenases immobilized on an ISFET.
- The developed biosensor effectively detects P450 monooxygenase activity, offering a sensitive electrochemical readout.
- The biosensor demonstrates applicability for the detection of chlorophenol compounds, paving the way for new environmental monitoring tools.