1 BioMEMS and Bionanotechnology: Integrated systems for Biology and Medicine Rashid Bashir Birck Nanotechnology Center, Discovery Park, School of Electrical and Computer Engineering, Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana http://engineering.purdue.edu/LIBNA July 17 th – 18 th , 2006 1 st Annual Conference Methods in Bioengineering, Cambridge, MA.
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1
BioMEMS and Bionanotechnology: Integrated systems for Biology and Medicine
Rashid Bashir
Birck Nanotechnology Center, Discovery Park, School of Electrical and Computer Engineering, Weldon School of Biomedical Engineering,Purdue University, West Lafayette, Indianahttp://engineering.purdue.edu/LIBNA
July 17th – 18th, 20061st Annual Conference Methods in Bioengineering, Cambridge, MA.
AcknowledgementsResearch Scientists/Post-docs:• Dr. Demir Akin• Dr. Amit Gupta• Dr. Jaesung Jang• Dr. Kwan Soup Lim
Graduate Students:• Hung Chang• Rameez Chatni• Angelica Davila• Oguz Elibol• Samir Iqbal• Yi-Shao Liu• Kidong Park• Bobby Reddy• Murali Venkatesan
BioVitesse, Inc. Co-Founder
Faculty Collaborators– Prof. A. Alam (ECE)– Prof. D. Bergstrom (Med Chem)– Prof. A. Bhunia (Food Science)– Prof. D. Peroulis (ECE)– Prof. M. Ladisch (Ag& Bio Engr) – Prof. G. Vasmatzis (Mayo Clinic)
Funding Agencies• NASA Institute on Nano-electronics and
Computing (INAC)• US Department of Agriculture (Center for
Food Safety Engineering at Purdue)• NSF, NSF NSEC at OSU• National Institute of Health, NIBIB• Discovery Park at Purdue University
• Use electrical & mechanical (optical) approaches at the micro and nanoscale in biochip sensors
• Label free, small sample size, array capability, cheap
DNACells ProteinsViruses
Molecules
8
Active Research Projects
http://engineering.purdue.edu/LIBNA
Glass cover
In/Out ports Cavities/ W ells
Epoxy adhesive
Pin 700µm
Lab-on-a-chip for Detection of Live
BacteriaLiu, Li, Gomez, Huang, Geng,
Bhunia, Ladisch, Bashir
Nano-Mechanical Cantilever Sensors for Detection of Viruses
Gupta, Akin, Broyles, Ladisch, Bashir
Nanopore Sensors for DNA DetectionChang, Andreadakis,
Kosari, Vasmatzis, Bashir
Dielectrophoresis Filters an Traps for Biological Entities
Li, Akin, Bhunia, Bashir
Electrical Characterization of DNA hybridization
Iqbal, Balasubramanian, Bergstrom, Bashir
Micro-Mechanical Cantilevers for
Detection of SporesDavila, Walter, Aronson,
Bashir
1um
Silicon Resistor
Silicon dioxide
Metal Contact Area
Au/Cr Electrode
Angle View with 85 degree (SEM, 10000x)
BASIC - Bio-inspired Assembly of
Semiconductor ICsLee, Bashir
Bacterial Mediated Delivery of Particles
in CellsAkin, Bashir
Trapping/Lysing of Bacteria/Viruses In
Microfluidic DevicesPark, Akin, Bashir
9
• Microfluidic devices as petri dish on a chip- “Impedance Microbiology” on a Chip- Hybrid Devices
• Electrical detection of spore germination• Nano-Cantilevers for virus and bacterial detection• Lysing of viruses in microfluidic devices• Nanopores for single DNA molecule characterization• Field effect device array for detection of proteins and
Pathogenic bacteria in foods is a serious concern to public healPathogenic bacteria in foods is a serious concern to public health: th: Approx. 76 million people get sick; 300,000 hospitalization, 5,0Approx. 76 million people get sick; 300,000 hospitalization, 5,000 death00 death
Needs for Rapid, Sensitive and Specific Needs for Rapid, Sensitive and Specific Detection Methods for BacteriaDetection Methods for Bacteria
(CDC, 2002(CDC, 2002
11
Technology Platforms for Rapid Detection of Live Bacteria
R. Bashir, ECE, BME A. Bhunia, Food Science M. Ladisch, BME, ABEJ. P. Robinson, BMS, BME
L. MonocytogenesOn a chip
• Rapid detection of live bacteria has wide applications– Industrial Microbiology (Pharmaceutical facility monitoring,
Food Safety)– Blood transfusion– Human Diagnostics– Global Health
• Rapid time to result limited by: – Amplification, enrichment, culture– 48-72 hours for viability determination and identification
• Center for Food Safety Engineering (www.cfse.purdue.edu) through cooperative agreement with USDA-ARS
P. P. Banada, Y. Liu, L. Yang, R. Bashir, and A. K. Bhunia, International Journal of Food Microbiology, 2006.
31
Overall Detection Scheme
Biotinylated Antibody (C11E9)
SiO2 surface
~14 nm
Streptavidin
Biotinylated BSA
~10 nm
~4 nm
Streptavidin
Biotinylated BSA
~10 nm
~4 nm
* Size information are obtained from Biochemistry 2nd
edition, R. H. Garrett and C. M. Grisham, 1995.
H. Li, et al., Transducers, 2005L. Yang, H. Li, D. Akin, T. Huang, A. Bhunia, M. Ladisch, R. Bashir, Lab Chip, 2006
• Antibody to L. Monocytogenes
– 150kD IgG– Binds to a 66kD
surface protein
32
DEP on DEP on
DEP off DEP off
104 ce
lls/5μl
103 ce
lls/5μl
104 ce
lls/5μl
103 ce
lls/5μl
DEP on
DEP off
Green - Listeria monocytogenes
0
5
10
15
20
25
30
35
Ab
capt
ure
effic
ienc
y
101 103102
27.1%
18.6% 19.1%
DEP captured cell number/5 μ l
0
5
10
15
20
25
30
35
Ab
capt
ure
effic
ienc
y
101 103102
27.1%
18.6% 19.1%
DEP captured cell number/5 μ l
Red - Enterobacter aerogenes & Enterococcus faecalis
At 103 / 2μl with a ratio of 1:1:1
C11E9 Antibody capture efficiency for
L. monocytogeneswith DEP
enhancement
33
BioVitesse, Inc.• ‘Petri dish on a chip’ to miniaturize impedance microbiology• To quickly and reliably detect and identify live bacteria in 2 to 4 hours, instead of 2 to 10 days• Provides in-process quality control monitoring systems• To the industrial microbiological market (Bio/Pharma and Food Safety)
Goal – Become the leader in rapid detection and identification of live cells
BioVitesse, Inc. Company Confidential
SiliconBiochip
Chip Cartridge
CCRTM
Cartridge
Automated System
Sample, Media,Sanitizing Fluid
34
Current Status and On Going Work
• Demonstrated automated electrical detection of 10-100cfu/ml in 100ml fluids in one time use CCR and Biochip cartridge
• Serial dilution and concentration• Integrated pH and impedance sensor• Antibiotic resistance of bacteria – drug screening• Slow growing bacteria (e.g. TB) & growth of bacteria in low O2
ambient (e.g. Obligate anaerobes)• Extraction of bacteria from blood and body fluids – medical
diagnostics• Nucleic acid based identification
35
Micro-fluidic Polymer Devices for Culture of Bacteria and Spores
Input TubeOutput Tube
PDMS Cover
SiliconOxide
MetalElectrodes
Bonding with PDMS
3-D fluidic Channel
Input TubeOutput Tube
PDMS Cover
SiliconOxide
MetalElectrodes
Bonding with PDMS
3-D fluidic Channel
Input Reservoir
in 3rd Layer of PDMS
1st Layerof PDMS
2nd Layerof PDMS
ChipUnderneath
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
0 5 10 15 20 25
Tim e (hours)
Impedance Magnitude [W
]
10:1, saturated10:1, unsaturated
2.5:1, unsaturated
2.5:1, saturated
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
0 5 10 15 20 25
Tim e (hours)
Impedance Magnitude [W
]
10:1, saturated10:1, unsaturated
2.5:1, unsaturated
2.5:1, saturated
Chang, et al., Biomedical Microdevices 5:4, 281-290, 2003,
Silicon Base, 3 PDMS layers, Top I/O port
36
Electrical Detection of Spore Germination
• Bacillus Anthracis spores (stern strain)• Alanine & Inosine as germinants• DPA is ~10 % of spore dry weight (~10-13 g)
-5
0
5
10
15
20
0 10 20 30 40
Elapsed Time (mins)
Con
duct
ivity
Cha
nge
(%)
Germinants Only
Spore Only 1e9 Cells/ mL
Spore with Germinants1e9 Cells/ml -
Macroscale Experiments
Spore
Bacteria
Inosine
Alanine
DPA(Dipicolinic
Acid)
Liu, et al. in preparation, 2006
37
Spore Germination in Chip
Spores labeled by OC63 and captured by DEP of electrodes
Conductivity vs. Time - 750 Hz1e10 Cells/mL
-5
15
35
55
0 20 40 60Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Conductivity vs. Time - 750 Hz1000 spores in 0.1nl = 1e10 Cells/ml
-5
15
35
55
0 20 40 60-5
15
35
55
0 20 40 60Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Spores wgerminant
Sporesonly
Conductivity vs. Time - 750 Hz1e10 Cells/mL
-5
15
35
55
0 20 40 60Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Conductivity vs. Time - 750 Hz1000 spores in 0.1nl = 1e10 Cells/ml
-5
15
35
55
0 20 40 60-5
15
35
55
0 20 40 60Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Spores wgerminant
Sporesonly
Conductivity vs. Time - 750 Hz1e9 Cells/ml
-10
0
10
20
0 20 40Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Conductivity vs. Time - 750 Hz100 spores in 0.1nl = 1e9 Cells/ml
-10
0
10
20
0 20 40Elapsed Time ( mins)
% C
hang
e of
|Con
duct
ivity
|
Spores wgerminant
Sporesonly
Comparison between Open & Close Valve Experiments(Open Valve: 700 Spores v.s. Close Valve: 100 Spores)
Whole Experiment v.s. Time
-5.00E-09
0.00E+00
5.00E-09
1.00E-08
1.50E-08
2.00E-08
Elapsed Time (minutes)
Net
cha
nge
inA
dmitt
ance
- 75
0 H
z
Close Valve - Germinant + Spore Open Valve - Germinant + Spore
Liu, et al. in preparation
• PDMS channels and valvesGlass base with metals
38
Integration of PDMS and Silicon
Transferable Microscale Metal Patterns for PDMS Metallization
(g)
(i)
(k)
(a)
Si waferAu layer
Ti layermercaptosilane layer
(h)
(j)(b)2nd Au (50 Å)
Ti (500 Å)1st Au (1500 Å)
Process flow of metal micro-patterning on PDMS: (a) substrate cleaning, (b) serial metal deposition (1500 Å of 1st Au, 500 Å of Ti, and 50 Å of 2nd Au) on Si wafer, (c) metal patterning with conventional photolithography, metal deposition (3000Å or 3rd Au), and lift off, (d) mercaptosilane treatment, (e) pouring and curing PDMS, f) peeling off PDMS from Si wafer, and (g) removal of 1st Au, Ti, followed by a short 2nd Au etch
Metal patterns are transferred to PDMS
Lim, et al. Lab Chip, 2006
39
Microscale Metal Patterns on PDMS
Minimum size of transferable pattern
40
BASIC: BIO-INSPIRED ASSEMBLY OF SEMICONDUCTOR ICs
Overall Approach:
Use of bio molecules and electro-hydrodynamics (EHD) for directed self assembly of silicon devices in 3-Dimensions
Indiana 21st Century R&T Fund
AC Electric
Field
Directed Assembly
Completed device released
in fluid
S. Lee, et al., Proceedings of MRS, 2001, 2002S. Lee, et al., Langmuir, April 2002
Molecules
41
3-D Heterogenous Integration
Displays/Electronics
Applications
• Processing temperature limits performance
– a-Silicon < 350°C– Poly-silicon < 650 °C– Single crystal < 1100 °C
• Single crystal on plastic !!– High resolution, flexible displays– Flexible Electronics
Lieber, et al, Feb 2nd, Sciencehttp://www.research.ibm.com/topics/popups/serious/nano/html/nhow.html
BASICC o m p lem en ta ry
m o lecu les
S e lf-A s sem b ly
D e vic e s
C o m p lem en ta rym o lecu les
S e lf-A s sem b ly
D e vic e s
42
Start Material(SOI Wafers)
Directed Self- Assembly
In Fluid
SOI NMOSFabrication
Device ReleaseIn Fluids
1st Layer Wafer 1st Layer Metal
Interconnect
1st SOI NMOS
M1 Layer
Open Contactsand Form 2nd
Device Layer
2nd Layer MetalInterconnect
2nd SOI PMOS
2nd SOI PMOS
M2 Layer
The processcan be repeated
over again tilldesired
3-Dimensional Integration
SOI PMOSFabrication
Device ReleaseIn Fluids
1st Layer Wafer
Open Contactsand Form 3rd
Device Layer
3rd SOI NMOS M2 Layer
S. Lee and R. Bashir, Advanced Materials, 2005
43
P-type silicon layer
Grown oxidePhotoresist
(c)
Silicon (100)
3-D MOSFET Body
(b)
Silicon (100)
(a)Highly doped S/D and gate regions
Nitride Spacer
(e)
(g)
Silicon (100)
Au/Cr S/D contact
Buried Oxide
(d)
Poly oxide feature Photoresist
Silicon (100)
Poly gate feature
Silicon (100)
(f)
Silicon (100)
Poly Silicon for gate
Silicon (100)
Grown poly oxide
Oxide for gate
(h)
Source
DrainGate
Transistor Process Flow
1,9-nonanedithiol
44
Transistor Release
S. W. Lee, R. Bashir, Advanced Materials, 2005
Oxide pillar
Silicon (100) (a)
45
Transistor Assembly
Randomly positioned
MOSFET in DI water
Assembled MOSFET in DI
water
Assembled MOSFET AF DI water was dried
completely
46
Electrical Results
Before release After Assembly
Rd, Rs ~ 0.5e4 Ω/μm2
47
Next Steps
• Array formation – flow through system or denser array
• Yield of release and assembly• 2-D and 3-D circuit demonstration• Orientation independent device
fabrication• Gate all-around device fabrication
48
BioMEMS Polymer/Silicon Cantilever Sensors
• Environmentally sensitive micro-patterned polymer structures on cantilevers
50 μm
Cantileverover a well
PMMA-based HydrogelPolymer
Z. Hilt, A. Gupta, O. Elibol, R. Bashir, N. Peppes
R. Bashir, J.Z. Hilt, A. Gupta, O. Elibol, and N.A. Peppas, Applied Physics Letters, Oct 14th, 2002; J. Z. Hilt, A. K. Gupta, R. Bashir, N. A. Peppas Biomedical Microdevices, September 2003, Volume 5, Issue 3, 177-184
- ΔpH = 1-10e-5 - pH = 6.5 ~ 1.9e5 H+ in 1000μm3
- ΔpH = 5e-4 change of ~ 150 H+
0
5
10
15
20
25
2.5 3.5 4.5 5.5 6.5 7.5 8.5
pH of Fluid Around the Cantilever
Def
lect
ion
at th
e tip
(μm)
Cantilevers touched the
bottom
ExperimentModel
slope = 18.3mm/pH(=1nm/5e-5ΔpH)
-
49
• Main aims:– Single molecule biophysics– Detection of hybridization – Towards direct sequencing of
single molecules
• Miniature DNA analyzer with ultra high sensitivity – Transduction events at single
molecule, nanoscale– Interface to the macroscale
Nanopore Channel Sensor Array for Characterization of Single Molecules
50
Fabrication Process
40
50
60
70
80
90
100
22500 23000 23500 24000 24500 25000Time (msec)
Cur
rent
(pA
)70
75
80
85
90
95
23144 23146 23148 23150 23152 23154
Time (msec)
Cur
rent
(pA
)
Pulses due to passage of dsDNA
Ox 1000 ASi 1400 AOx 4000 A
Handle layer
Ox 1000 A
Open submicron etch window on front side by e-beam lithography
Handle layer
Ox 1000 ASi 1400 AOx 4000 A
Ox 1000 A
Etch through the SOI wafer from backside
Ox 1000 ASi 1400 AOx 4000 A
Handle layer
Do front side etch to make a nanopore on buried oxide layer
50 – 60 um
Cont’d
Si1400 AOx 4000 A
Handle layer
Remove buried oxide layer to open a nanopore
Si1400 A
Initial opened pore is around 50-100nm
50-100nm
230-240 nm
2-4nm
Do thermal oxidation and TEM processing toshrink pore to less than 5 nm
H. Chang, F. Kosari, G. Andreakakis, M. A. Alam, G. Vasmatzis, R. Bashir, Nano Letters, 2004.H. Chang, S. M. Iqbal., E. A. Stach, A. H. King, N. J. Zaluzec, R. Bashir, , Applied Physics Letters, 2006.
~50nm Silicon
100nm SiO2
100nm SiO2
~36 nm
Not drawn to scale
200bp DNABuffer solution : 0.1 M KCl, 2 mM Tris(pH 8.5)Ag/AgCl electrodesBias : 200 mV
Loaded resonant frequency = 658 kHz Unloaded resonant
frequency = 664kHz
In air
Sensitivity - 20um long cantilevers
y = 3.6548x - 1.6627R2 = 0.9236
0
5
10
15
20
25
30
0 2 4 6 8 10
Number of B.anthracis spores
Dow
nwar
d Fr
eque
ncy
Shift
(k
Hz)
60A. Gupta, D. Akin, R. Bashir, Applied Physics Letters, 2004.L. Johnson, A. Gupta, A. Ghafoor, D. Akin, R. Bashir, Sensors and Actuators, 2006
Mass Detection of Virus Particles
f0 = 1.27 MHzf1 = 1.21 MHz
Q ~ 5k = 0.006 N/m
Δf=60kHz
• Ultimate Sensitivity ~ 6.3 Hz/ag (in air)• Min. Detectable Mass (Q = 5, Δf = 40kHz) ~ 6.3fg• Min. Detectable Mass (Q = 500, Δf = 400Hz) ~ 50ag• Work on going to integrated concentration elements• Integrate Abs on surface – in humid air !
Work featured in Popular Science, EE Times, Work featured in Popular Science, EE Times, Nature News, and CNBC TV ShowNature News, and CNBC TV Show
61
Effects of Viscous Medium
• Resonant frequency decreases• Quality factor decreases• Sensitivity decreases
Unloaded air resonant
frequency = 548.6 kHzUnloaded fluid resonant
frequency = 121.3 kHzQ = 1.8
Q = 36.3
Q =
4μπρb2 + Γr ωR( )
Γi ωR( )
– μ = mass per unit length– ρ = density of the medium– b = width of the cantilever– Γr(ωR) and Γi(ωR) are the real and imaginary parts of the hydrodynamic function Γ(ω), with ωR as the resonant frequency
Y. Martin et al., J. Appl. Phys., 61, 1987T.Braun et al., Phys. Rev. E 72, 2005T. R. Albrecht et al., J. Appl. Phys., 69, 1991
01*
2 c l
kfm mπ
=+
– k = spring constant– mc = mass of cantilever– ml = mass of liquid
• Driving the cantilevers can reduce this down to ~ 10-20pg
Sensitivity - 20μm length
y = 0.1989x - 0.8316R2 = 0.82
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140 160
No. of B.anthracis spores
Freq
uenc
y sh
ift (k
Hz)
63
Final Remarks• Integration of various materials and functions on a chip• Concentration, specific capture, growth, lysing detection• Applications & fundamental biophysics• High end label free sensors that are top down fabricated
• Integrated Biochips : Sample Rapid Results• Micro-environments for single cell, cell-cell analysis, cellular
surgery and manipulation• Implantable devices for integrated diagnostic and therapeutic
devices
64
Thank You
65
Target Concentrations and Detection Limits
• Food Safety:– FDA has zero tolerance: <1cfu/100-250ml for some pathogens (e.g.
Listeria Monocytogenes)– Various limits from 1cfu/ml to 100cfu/ml at different points in
Flow channel and output aerosol collection chamber
Cantilever chip
• The electrospray aerosol generator can produce neutralized, monodisperse, nano aerosols including viruses or proteins.
• The flow and chip channel are designed to maximize the particle transport to the chip and optimize flow velocity around the cantilevers to capture viruses with DEP forces.
Flow direction
68
25 mm filter holder25 mm filter holder
47 mm filter holder47 mm filter holder
Air SpaceAir Space
CCR KiT Assembly
Uses membranes in series to process 100 mL hot dog extract into 0.1 to 1 mL sample
Ladsich, et al. USDA Review, 2004
69
Mech/Elect.Detection
DNA, protein
Cantilevers,NanoFETs, Nano-pores
Cell Lysing
Nano-probeArray
Micro-scaleImpedance
Spectroscopy
ViabilityDetection
Conc.Sorting
On-chipDielectro-phoresis
Fluidic Ports
Integrated Systems for Study ofCells and Microorganisms
SelectiveCapture
Ab-based
Capture
“Lab on a Chip” Enabled by BioMEMS and
Bionanotechnology
MEMSFilters
Filters
70
Detection of Listeria Cells
Frequency change after binding of 180 dry bacteria cells
Unloaded Resonant Frequency (85.6 kHz)
Loaded Resonant Frequency (84.7 kHz)
6.5 7 7.5 8 8.5 9.59 10x 104Frequency (Hz)
Ampl
itude
(uni
ts)
10-11
10-10
10-9
10-8
Individual Listeria innocuabacterial cells
10 μm1 μm
Non-specific binding of Listeria innocua bacterial cells to a cantilever beam
Lxm* = (x/L)m
A. Gupta, D. Akin, R. Bashir, JVST-B, 2004.Craighead, et al. APL, 77, 3, 17th July 2001, 450-452
ωo2 = k
m
71
Use of Antibodies
• Spores drift in fluid• Antibodies used to immobilize
spores onto the surface• Bovine serum albumin (BSA) used
to prevent non-specific capture• Antibodies for specific capture
Cantilever
Antibodies
Spores
10μm200X 10μm200X
No antibody
With antibody
Before Measurements After Measurements
72
Sensing Methods in BioChips
Surface Stress Change Detection
Δz
L
t
• Δz = deflection of the free end of the cantilever• L = cantilever length• t = cantilever thickness• E = Young’s modulus• ν = poison’s ratio• Δσ1 change in surface stress on top surface• Δσ2 change in surface stress on bottom surface
( ) ( )21
2 14 σσνΔ−Δ
−⎟⎠⎞
⎜⎝⎛=Δ
Etlz
Surface Stress Change Detection
Δz
L
t
• Δz = deflection of the free end of the cantilever• L = cantilever length• t = cantilever thickness• E = Young’s modulus• ν = poison’s ratio• Δσ1 change in surface stress on top surface• Δσ2 change in surface stress on bottom surface
( ) ( )21
2 14 σσνΔ−Δ
−⎟⎠⎞
⎜⎝⎛=Δ
Etlz
⎟⎟⎠
⎞⎜⎜⎝
⎛−=Δ 22
12
114 off
kmπ
mkf
π21
=
• k = spring constant• m = mass of cantilever• f0 = unloaded resonant frequency• f1 = loaded resonant frequency
Mass Change Detection
⎟⎟⎠
⎞⎜⎜⎝
⎛−=Δ 22
12
114 off
kmπ
mkf
π21
=
• k = spring constant• m = mass of cantilever• f0 = unloaded resonant frequency• f1 = loaded resonant frequency