Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 312

CHAPTER EIGHT

Fluorescent Protein-Based
Biosensors and Their Clinical
Applications

Yusuke Ohba * , Yoichiro Fujioka * , Shigeyuki Nakada † , Masumi Tsuda *
*Laboratory of Pathophysiology and Signal Transduction, Hokkaido University Graduate School of Medicine,
N15W7, Kita-ku, Sapporo, Japan
† Mitsui Engineering and Shipbuilding Co. Ltd., Tamano Technology Center, 3-16-1 Tamahara, Tamano,
Okayama, Japan

Contents
1.

Introduction 314

2. Fluorescent Proteins 315

2.1 Chromophore formation of GFP and its derivatives 315

2.2 Red chromophore synthesis via blue intermediates 317

2.3 Circular permutation 318

2.4 Fluorescent proteins as tags 318

2.5 Spectral unmixing (compensation) for multicolor imaging 319

3. Single-Fluorescent Protein-Based Biosensors 321

3.1 Fluorescent timers 321

3.2 Photoactivatable fluorescent proteins 322

3.3 Sensor for ions and small molecules 322

4. Bimolecular Fluorescence Complementation 323

4.1 Advantages of BiFC 325

4.2 Disadvantages of BiFC 325

5. Förster Resonance Energy Transfer 326

5.1 Intermolecular (or bimolecular) FRET 328

5.2 Intramolecular (or unimolecular) FRET 330

6. Methods for Evaluating FRET Efficiency 332

6.1 Donor fluorescence lifetime as a measure of FRET efficiency 333

6.2 Time domain 334

6.3 Frequency domain 334

6.4 Lifetime measurement using a flow cytometer 335

7. Clinical Application of FRET 337

7.1 Biosensor design and its specification 338

7.2 Special features of Pickles 339

7.3 Evaluation of drug efficacy in patient-derived cells 340

7.4 Future tasks 342

8. Closing Remarks 342

References 343