In Situ Detection of Chromogranin A (CgA) Released from Living Neuron Using Single-Walled Carbon Nan...
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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

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


Scientific collaborators

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


Outline

Outline

  • Motivation

  • Introduction to NW/NT-FET biosensor

  • Fabrication of SWNT-FET

  • Experimental setup

  • Molecular recognition and detection of CgA

  • Summery


Scientific collaborators

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


Scientific collaborators

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


Scientific collaborators

II. Introduction to NW/NT-FET biosensor

NW/NT-FET sensor

MOSFET

Yi Cui, Charles M. Lieber, Science. 293, 1289


Scientific collaborators

Antibody

Antigen

Source

Drain

Blocking agent

Linker

NW/NT

p-Si backgate

II. Introduction to NW/NT-FET biosensor


Scientific collaborators

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


Iii fabrication of swnt fet

III. Fabrication of SWNT-FET

welding

Microfludic channel

Chii-Dong Chen Lab. Institute of Physics, Academia Sinica


Sem image of swnt fet

I – Vg plot

p-type

III. Fabrication of SWNT-FET

SEM image of SWNT-FET

Electrode

SWNTs

500 nm


Iv experimental setup

IV. Experimental setup

GPIB

Lock in amplifier

SR830, EG&G 7465

SWNT-FET

VSD supply

GPIB

Microfludic

channel

Syringe pump

Keithley 2400

VG supply


Chemical modification

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.


Primary neuronal cell culture

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


Scientific collaborators

IV. Experimental setup

(b)

(c)

(a)

10 μm

Soma

Neurite

10 μm

(b)

  • Immunochemistry

  • Western blot

(c) Immobilization of CgA-Ab


Scientific collaborators

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 :


Molecular recognition of cgap and cga ab

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


Scientific collaborators

V. Molecular recognition and detection of CgA

Synaptic transmission

vesicle

receptor


Scientific collaborators

V. Molecular recognition and detection of CgA

In-situ detection of CgA released by neuronal cells


Vi summery

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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