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 Surface Plasmon Resonance
> 1 fM (antibody)
Nature Protocols 2007
Cantilevers Sensor
< 50 pM (DNA)
Nature 2007, 446, 1066 - 1069
< 1 pM (streptavidin)
Nature Reviews Drug Discovery 2002, 1, 515-528
Nanowire Field-effect Transistor
I
< 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
Character.
Commercial available
Detection limits*
Portable?
CMOS compatible
Detection
Schemes
Micro & Nano
Cantilevers
No <50 pM Yes/No Yes
Response time
Fast
Surface
Plasmon
Resonance
ELISA
Si Nanodevices
Yes
Yes
No
< 1 pM
>1 fM
<1 fM
No
No
Yes
No
No
Yes
Very
Fast
Slow
Vary fast
* Based on published data on SCI articles.
Operation Principle of SiNW-FET
Biosensors
+ +
+
S D S D
BOX BOX
G (I/V)
G
0
G (I/V)
G
0
Δ G
Time (s) Time (s)
Silicon nanowire field effect transistor (SiNW-
FET)
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 Bottom-up: VLS
James R. Heath (Caltech) Charles M. Lieber (Harvard)
Nanolett., 2006 J. Am. Chem. Soc. 2000
Our strategy
• Silicon-based nanowires or nano-belt
• Compatible with current industrial process
Spacer Lithography Technology
Etch
Removal of sacrificial
Layer and etch
E-beam lithography
SiNW
Spacer lithography
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
PDMS m-
Fluidic
Channel
SiNWs
After UV/Ozone treatment, sample and microfluidic channel were bonded.
1 cm
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-
K
eto
S
teroid
I
somerase (
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
HN
H
N
O
I
SO
3
H
HN
H
N
O
I
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
NH
SO
3
H mA51
H
N
O
SH
19-NT mA51
Design of a Nano-device for Steroids Detection steroids
Protein design
Detection of Non-charge
Analytes on SiNW-FET
Reporting molecule (probe)
Fabrication and modification of
SiNWs
Gene construction
&
Overexpression
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-NH
2
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-ETHYL-
17β-OL-3-ONE
Steroids
19-Norandrostendinone
Norethandrolone
Progesterone
Testosterone
17α-Estradiol
K d
, μM
10.9
2.0
1.9
76
27
PROGESTERONE
19-NORANDROSTENDIONE
17α-ESTRADIOL
TESTOSTERONE
∆ m/z = 367 Da
MW = 367 Da m/z of KSI_A51
[mod_A51] : [KSI] = 1:1
In pH 7.6 Tris – HCl buffer
For 24 hrs.
m/z of KSI
Direct Immobilization of Enzyme onto the Si Nanowire Surface
NH
2
O
OH
S O
O
N
O
O
O
O
OH
S O
O
N
O
O
KSI
NH
O
O KSI
OH OH OH
Si
O O
O
NH
2
NH
2
NH
2
O
O
N
O
O
O S O
OH NH
2
HN
O
NH
2
NH
2
HN
O
NH
2
SiO2
NH
2
APTS
SiO2
BS3
Sulfo-NHS ester
SiO2
KSI-126C
KSI-126C
SiO2
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.
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