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Scientific collaborators

In Situ Detection of Chromogranin A (CgA) Released from Living Neuron Using Single-Walled Carbon Nanotube Field Effect Transistors C. C. Tsai ( 蔡佳璋 ) Institute of Atomic and Molecular Sciences, Academia Sinica 2006-07-27. Scientific collaborators.

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Scientific collaborators

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  1. In Situ Detection of Chromogranin A (CgA) Released from Living Neuron Using Single-Walled Carbon Nanotube Field Effect TransistorsC. C. Tsai (蔡佳璋)Institute of Atomic and Molecular Sciences, Academia Sinica2006-07-27

  2. Scientific collaborators Chen-Wei Wang,1 Chien-Yuan Pan (潘建源),2,* Hsing-Chen Wu,1 Po-Yuan Shih,2 Kuo-Tang Liao,3 Li-Long Lu,2 Chii-Dong Chen (陳啟東),4,* and Yit-Tsong Chen (陳逸聰)1,3* 1. Department of Chemistry, National Taiwan University 2. Department of Life Science and Institute of Zoology, National Taiwan University 3. Institute of Atomic and Molecular Sciences, Academia Sinica 4. Institute of Physics, Academia Sinica

  3. Outline • Motivation • Introduction to NW/NT-FET biosensor • Fabrication of SWNT-FET • Experimental setup • Molecular recognition and detection of CgA • Summery

  4. I. Motivation Chromogranin A (CgA) • Acidic secretory protein, pI ~ 4.5 • Molecular Weight ~ 50 k Da • ＣgA is very abundant in neuron and neuronendocrine cells and is co-released with neurotransmitters during the synaptic transmission process. => CgA level released by neuronal cells can refer to the neuronal activity. Medical application: 1. Neuroendocrine tumor 2. Neurodegenerative disease

  5. I. Motivation Advantage of NW/NT-FET: • High sensitivity (theoretical limit = 1 fM) • High selectivity • Label-free detection • In-situ detection Recent research goal: • Improving the performance • Medical application : virus, cancer marker, and Hereditary hemochromatosis. Paul E. Sheehan and Lioyd J. Whitman. Nano Lett, 2005, 5, 803 Jong-inHahm, Charles M. Lieber, 2004, Nano Lett, 4, 51 Fernando Patolsky, Charles M. Lieber, 2004, PNAS,101,14017 Gengfeng Zheng, Charles M. Lieber, Nat. Bio, 2005, 23,1294 Alexander Star, and Christian Valcke, PNAS, 2006, 103, 921

  6. II. Introduction to NW/NT-FET biosensor NW/NT-FET sensor MOSFET Yi Cui, Charles M. Lieber, Science. 293, 1289

  7. Antibody Antigen Source Drain Blocking agent Linker NW/NT p-Si backgate II. Introduction to NW/NT-FET biosensor

  8. III. Fabrication of SWNT-FET 1.5 cm 0.6 cm 2μm SWNTs 20 μm Chii-Dong Chen Lab. Institute of Physics, Academia Sinica Fernando Patolsky, Charles M. Lieber, 2004, PNAS,101,14017

  9. III. Fabrication of SWNT-FET welding Microfludic channel Chii-Dong Chen Lab. Institute of Physics, Academia Sinica

  10. I – Vg plot p-type III. Fabrication of SWNT-FET SEM image of SWNT-FET Electrode SWNTs 500 nm

  11. IV. Experimental setup GPIB Lock in amplifier SR830, EG＆G 7465 SWNT-FET VSD supply GPIB Microfludic channel Syringe pump Keithley 2400 VG supply

  12. IV. Experimental setup Chemical modification 1-pyrenebutanoic acid, succinimidyl ester tween20 Steric effect π-π stacking hydrophobic interaction R.J. Chen, Y. Zhang, D. Wang, H. Dai. J. Am. Chem. Soc. 2001, 123, 3838. R. J . Chen, H. Dai, Proc. Nat. Acad. Sci. 2003, 100, 4984.

  13. IV. Experimental setup Primary neuronal cell culture Embryo Midbrain Soma Group of soma Brain Neurites Neurites Chien-Yuan Pan Lab, Department of Life Science and Institute of Zoology, National Taiwan University 100 μm 50 μm

  14. IV. Experimental setup (b) (c) (a) 10 μm Soma Neurite 10 μm (b) • Immunochemistry • Western blot (c) Immobilization of CgA-Ab

  15. V. Molecular recognition and detection of CgA Sensing mechanism Chemical gating effect： Since the low isoelectric point of CgAP (pI ~ 4.5), CgA is negatively charged in PBS buffer at pH = 7.4. p-type vs. CgA Thining the Schottky barrier ：

  16. V. Molecular recognition and detection of CgA Molecular recognition of CgAP and CgA-AB 100 nM CgAP 4 ﹪up 10 nM CgAP 60μ g/mL BSA 1 nM CgAP 0.7 ﹪up

  17. V. Molecular recognition and detection of CgA Synaptic transmission vesicle receptor

  18. V. Molecular recognition and detection of CgA In-situ detection of CgA released by neuronal cells

  19. VI. Summery • From the molecular experiment of CgAP and its antibody, we achieve the detection limit ~ 1nM, which is sufficient for typical diagnosis of neuroendocrine tumor (~ 2 nM). The sensing mechanism was thought to be the combination of chemical gating effect and Schottky barrier thining. • Our study has demonstrated that CgA released from the synaptic terminal of neurons can be detected directly with high selectivity and sensitivity by CgA-Ab modified SWNT-FET. • This sensing technique can further be applied to study the activity of an individual neuron, which should open a new window to understand the neurophysiology in neuronal network.

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