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. 2011;6(7):e22382.
doi: 10.1371/journal.pone.0022382. Epub 2011 Jul 27.

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells

Yi-Chun Kuo  1 , Jennifer H Ho, Ta-Jen Yen, How-Foo Chen, Oscar Kuang-Sheng Lee
Affiliations

Development of a surface plasmon resonance biosensor for real-time detection of osteogenic differentiation in live mesenchymal stem cells

Yi-Chun Kuo et al. PLoS One. 2011.

Abstract

Surface plasmon resonance (SPR) biosensors have been recognized as a useful tool and widely used for real-time dynamic analysis of molecular binding affinity because of its high sensitivity to the change of the refractive index of tested objects. The conventional methods in molecular biology to evaluate cell differentiation require cell lysis or fixation, which make investigation in live cells difficult. In addition, a certain amount of cells are needed in order to obtain adequate protein or messenger ribonucleic acid for various assays. To overcome this limitation, we developed a unique SPR-based biosensing apparatus for real-time detection of cell differentiation in live cells according to the differences of optical properties of the cell surface caused by specific antigen-antibody binding. In this study, we reported the application of this SPR-based system to evaluate the osteogenic differentiation of mesenchymal stem cells (MSCs). OB-cadherin expression, which is up-regulated during osteogenic differentiation, was targeted under our SPR system by conjugating antibodies against OB-cadherin on the surface of the object. A linear relationship between the duration of osteogenic induction and the difference in refractive angle shift with very high correlation coefficient was observed. To sum up, the SPR system and the protocol reported in this study can rapidly and accurately define osteogenic maturation of MSCs in a live cell and label-free manner with no need of cell breakage. This SPR biosensor will facilitate future advances in a vast array of fields in biomedical research and medical diagnosis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. SPR setups and experimental procedures.
(A) Schematic illustration of the SPR device: The He-Ne laser penetrated through a polarizer and a beam splitter, which split the beam 50/50. One was detected by a photodiode and the other one was coupled by a prism to generate Surface Plasmon Wave on the Au chip in which the angle shift was detected by a photodiode. (B) The OB-cadherin expressing cells flowing into the chamber were captured by the Au chip pre-coated with OB-cadherin antibodies, which changed the angle of the reflected laser beam. (C) A typical graphical data from SPR measurements.
Figure 2
Figure 2. Molecular biology detection of MSC-osteoblast differentiation.
(A) Real-time PCR of OB-cadherin expression. (B) Western blot of OB-cadherin expression during osteogenic induction of MSCs. SaOS2 served as positive control. (C) The OB-cadherin expression level intensity analyzed by semi-quantitative method and was normalized to the intensity of SaOS2. (D) Alkaline phosphatase (AP) staining and von kossa (VK) staining.
Figure 3
Figure 3. Resonance characteristics of antibody–antigen reaction on Au coated chips.
(A) Test control of antibody non-coating system and injected with SaOS2, Hep3B and MSCs onto Au chip. (B) Test control of antibody coating system by injecting SaOS2, Hep3B and MSCs onto mouse IgG pre-coated Au chip. (C) The verification and stability test of OB-cadherin antibody-antigen binding by injecting SaOS2, Hep3B and MSCs.
Figure 4
Figure 4. Angle shift difference during osteogenic differentiation of MSCs.
(A) The angle difference, formula image, as a function of culture time. (B) OB-cadherin expression normalized to SaOS2 during differentiation.
See this image and copyright information in PMC

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