Transcript SiNW-FET

Bio-recognition and Bio-sensing
本研究與奈米所許鉦宗教授研究室合作完成
The Potential and Niche of Biosensors
• The global market for biochips increased from $1,637.5
millions in 2006 to $2,115.6 millions by the end of 2008.
• It should reach $3,840.7 millions in 2013, a compound
annual growth rate (CAGR) of 12.7%.
Basic Structure of Biosensor
Bio-recognition
Interface
Energy transducer
Signal
processing &
data output
Techniques for transduce
• Surface Plasma Resonance
• Cantilever sensor
• Field effect transistor
• others
Biomolecule Detection Methods
Enzyme-linked Immunosorbent Assay
> 1 fM (antibody)
Nature Protocols 2007
Cantilevers Sensor
Surface Plasmon Resonance
< 1 pM (streptavidin)
Nature Reviews Drug Discovery 2002, 1, 515-528
Nanowire Field-effect Transistor
I
< 50 pM (DNA)
Nature 2007, 446, 1066 - 1069
< 1 fM (DNA, antibody)
Nature 2007, 445, 519-522
Good for big molecule detection!!
Yet, the detection of small
molecule is a great challenge!!
Comparison of Biosensing Techniques
Response
Character. Commercial Detection Portable?
CMOS
time
available
limits*
compatible
Detection
Schemes
Micro & Nano
Cantilevers
No
<50 pM
Yes/No
Yes
Fast
Surface
Plasmon
Resonance
Yes
< 1 pM
No
No
Very
Fast
ELISA
Yes
>1 fM
No
No
Slow
Si Nanodevices
No
<1 fM
Yes
Yes
Vary fast
* Based on published data on SCI articles.
Operation Principle of SiNW-FET
Biosensors
+ +
+
S
S
D
BOX
D
BOX
G (I/V)
G (I/V)
G0
G0
ΔG
Time (s)
Time (s)
Silicon nanowire field effect transistor (SiNWFET)
B
Yi Cui, Qingqiao Wei, Hongkun Park, Charles M. Lieber, Science (2001) 293, 1289.
Proc Natl Acad Sci U S A. 2005,102, 3208–3212.
Single virus detection
Nanomedicine 1(1), 51, 2006
World Leading Groups
Top-down: Nanoimprint
James R. Heath (Caltech)
Nanolett., 2006
Bottom-up: VLS
Charles M. Lieber (Harvard)
J. Am. Chem. Soc. 2000
Our strategy
• Silicon-based nanowires or nano-belt
• Compatible with current industrial process
E-beam lithography
SiNW
Etch
Removal of
sacrificial
Layer and
etch
SiNW Resist
Resist
Spacer Lithography Technology
Spacer lithography
SiNW
SiNW
Array of Silicon Nanowire FETs
SU-8 Microfluidics
• Sidewall spacer
technique
S
D
D
S
Dimensions: L > 1 μm, H = 20~70 nm, W > 40 nm
Integration of SiNW-FETs with PDMS
microfluidic Channel for Analytes Delivery
PDMS microfluidics
Sample size
SiNWs with Pads
SiNWs
PDMS
mFluidic
Channel
1
cm
After UV/Ozone treatment, sample and
microfluidic channel were bonded.
Chips bonded onto a PCB board for
measurement
The established biosensing platform---On the way for
miniaturization
How to overcome the intrinsic
limitation of SiNW-FET?
To detect a non-charged small
molecule such as steroids.
The importance of steroids analysis
1. Estrone (E1) and estradiol (E2)
determine the risk of breast cancer
2. Restricted drugs in sports
3. Hormone.
17α-ESTRADIOL
TESTOSTERONE
Idea
△5-3-KetoSteroid Isomerase (KSI)
(1) Gene has been cloned and overexpressed.
(2) A dimeric enzyme (m.w. 13.4 kDa/sub-unit) with a
hydrophobic cavity containing Asp-38 and Tyr-14 as
the essential groups.
(3) X-ray protein structure has been solved.
(4) Strong affinity toward steroids
Fluorescence of I14-labeled KSI and I14 in IPA
H
N
H
N
I
HN
O
O
SO 3 H
I
HN
SO 3 H
I14
Isolation of I14-labeled peptides
MS/MS analysis of I14-labeled KSI
(a) The result of MS/MS of M/Z = 822.7 (2+) was shown
(b) The result of MS/MS of M/Z = 975.8 (2+) was shown
∆ m/z = 306 ( I14 – I )
Asp-38
Tyr-14
S O 3H
mA51
H
N
SH
NH
O
19-NT
mA51
Design of a Nano-device for Steroids Detection
steroids
Detection of Non-charge
Analytes on SiNW-FET
Protein design
Reporting molecule (probe)
Gene construction
&
Overexpression
Fabrication and
modification of
SiNWs
SiNWs
Protein modified by probe
protein
immobilization
Improve and Optimize
protein function
Detection system
Experimental Design for Chemical
modification
Steps involving
chemical reaction
Cys : R-SH
Link to reporter
Lys : R-NH2
Link to SiNWs
Y55F/K60R/F86C/Y88G/K92R/K108R/K119R/A125K
Construction and Purification of KSI
Dissociation constants of
Steroids with KSI
4-ESTREN-17α-ETHYL17β-OL-3-ONE
Steroids
Kd, μM
19-Norandrostendinone
10.9
Norethandrolone
2.0
Progesterone
1.9
Testosterone
76
17α-Estradiol
27
PROGESTERONE
19-NORANDROSTENDIONE
17α-ESTRADIOL
TESTOSTERONE
MW = 367 Da
m/z of KSI_A51
∆ m/z = 367 Da
m/z of KSI
[mod_A51] : [KSI] = 1:1
In pH 7.6 Tris – HCl buffer
For 24 hrs.
Direct Immobilization of Enzyme onto
the Si Nanowire Surface
OH
O S O
O
OH
OSO
N
NH2
O
KSI
O
O
O
O
N
O
NH
O
O
O
KSI
O
Si
O
O
O
O
N
O
OH
OH
NH2
OH
NH2 NH2
O S O
OH
SiO2
SiO2
APTS
NH2
O
O
NH2 HN
NH2
NH2
HN
SiO2
BS3
Sulfo-NHS ester
NH2
SiO2
KSI-126C
KSI-126C
Citrate-passivation gold nanoparticles were deposited onto the KSI-modified surface
Real-time Sensing on Steroid
Linear correlation 1 fM~0.1 pM
Conclusion
We demonstrated the design, fabrication and
testing of the world’s first SiNW-FET device
for non-charged steroid detection with the
sensitivity greater than 10-16 M.