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Introduction to BioMEMS & BionanotechnologyLecture 4

R. BashirR. BashirLaboratory of Integrated Biomedical Micro/Nanotechnology and

Applications (LIBNA), Discovery ParkSchool of Electrical and Computer Engineering,

Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana

http://engineering.purdue.edu/LIBNA

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Key Topics

• Biochips/Biosensors and Device Fabrication• Cells, DNA, Proteins• Micro-fluidics• Biochip Sensors & Detection Methods • Micro-arrays• Lab-on-a-chip Devices

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Micro-fluidic Devices for Conductance Detection of Bacterial Metabolism

• Detection of Cell Growth by measuring their metabolic activity in micro-fluidic devices

R. Gomez, et al., Biomedical Micro-Devices, vol. 3, no. 3, p. 201-209, 2001.R. Gomez, et al., Sensors and Actuators, B, 86, 198-208, 2002.

ZwZw ZwZw

Cdi

Rs

Dielectric capacitance

Electrolyteresistance

Electrode-electrolyte interfaces

Electrode-Electrolyte

Interface Model:

Constant-angleimpedance

BjZ nw )(

1ω

=

4

Target species

cell

Controlled Micro-culture Environment On a Chip

(temperature, O2, CO2, H2O)

Electrical signal

Optical signalTarget

species

cell

Controlled Micro-culture Environment On a Chip

(temperature, O2, CO2, H2O)

Electrical signal

Optical signal

4. Cell-Based Sensors/Biochips

L. Bousse, Whole cell biosensors, Sensors and Actuators B (Chemical), Vol. B34, No. 1-3, August 1996, pp. 270-5.J.J. Pancrazio, J.P. Whelan, D.A. Borkholder, W. Ma, D.A. Stenger, Development and application of cell-based biosensors, Annals of Biomedical Engineering, Vol. 27, No. 6, November 1999, pp. 697-711.D.A. Stenger, G.W. Gross, E.W. Keefer, K.M. Shaffer, J.D, Andreadis, W. Ma, J.J. Pancrazio, Detection of physiologically active compounds using cell-based biosensors, Trends in Biotechnology, Vol. 19, No. 8, August 1, 2001, pp. 304-309.

• The transductions of the cell sensor signals maybe achieved by:– the measurement of

transmembrane and cellular potentials,

– impedance changes, – metabolic activity, – analyte inducible emission of

genetically engineered reporter signals, and

– optically by means of fluorescence or luminescence.

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5. Micro/Nano-scale Coulter Counter

I

t

+

-

I

t

I

t

Cis chamber

Trans chamber

----

----

----

6Cross section of

micro-fabricated pore

Micro-pore for cellular studies

• Micro-devices for single cell characterization – utilize the charge properties

• Micro-fabricate a pore where single entity can pass

- -

+ve Voltage+ve Voltage

- -------

--

Si

SiO2

Micro-pore

Optical Picture of a Pore in a

micro-fabricated filter

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Microscale Coulter Counter Velocity (cm/s) vs. Electrical Field (V/cm)

Live Listeria innocua with pore

v = -5e-7E - 0.007; R2=0.814

0E+00 0.5E-04 1.0E-03 2.0E-03 2.5E-03 3.0E-03 3.5E-03 4.5E-03

-2.1E+04 -1.8E+04 -1.5E+04 Electrical Field (V/cm)

Vel

ocity

(cm

/s) Mobility = -5e-7 cm2/V-s

I-T Diagram for Live Listeria, 1e8/ml, V = 40 V, 05112010

1.25E-05

1.30E-05

1.35E-05

1.40E-05

1.45E-05

1.50E-05

1.55E-05

1.60E-05

1.65E-05

1.70E-05

35 40 45 50 55

Time (s)

Cur

rent

(A)

Live Listeria innocuawith a well-defined

cell wall

H. Chang, A. Ikram, T. Geng, F. Kosari, G. Vasmatzis, A. Bhunia, and R. Bashir, “Electrical characterization of microorganisms using microfabricated devices”, Journal of Vacuum Society and Technology B, 20, 2058 (2002).

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a-hemolysin nanochannel

The model of DNA passing through an a-hemolysin channel.

- a-hemolysin channel, a biological protein based-pore, was utilized.- Pore size is 2.6 nm.- Both RNA and DNA molecules were observed traversing the nanochannel.

Poly-C

Poly-A

Nanoscale DNA Coulter Counter

Kasianowicz et al., 1996, Meller, et. al. 2000.

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- Solid-state based nanopore. Made in silicon nitride membrane. - Pore size: 3 nm and 10 nm.- The relation among DNA lengths and translocation times and applied biases were determined.

Fabrication Techniques

TEM of Li’s nanopore. b. DNA measurement setup in Li’s work. From Li et. al. Nature

Materials, 2003

The fabrication of Li’s nanopore. From Li et. al. Nature, 2001.

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DNA Translocation

Current fluctuations when DNA was passing through the pore

Histograms of relation among DNA lengths, translocation times and

applied biases.

Li et. al. 2003

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Silicon Based Nanopore(Not to scale)

Start with a (100) 4 inch SOI wafer. Thickness : 525 um. SOI : 250 nm,

Buried oxide layer: 400 nm.

Si 2500 AOx 4000 A

Handle layer

Start with a (100) 4 inch SOI wafer. Thickness : 525 um. SOI : 250 nm,

Buried oxide layer: 400 nm.

Si 2500 AOx 4000 A

Handle layer

1.

Grow thermal oxide on wafer surface and open etch window to etch through the

handle layer. Etch stops on buried oxide layer.

Ox 4000 A

Ox 1000 ASi 1400 A

2.

Remove buried oxide layer and regrow 100 nm thermal oxide.

30-40 nm

Remove buried oxide layer and regrow 100 nm thermal oxide.

30-40 nm4.

On SOI layer, open another etch window to etch through the SOI layer. Etch stops on

buried oxide layer.

70-80 nm

230-240 nm

Si 1400 A

Ox 4000 A

Ox 1000 A3.

On SOI layer, open another etch window to etch through the SOI layer. Etch stops on

buried oxide layer.

70-80 nm

230-240 nm

Si 1400 A

Ox 4000 A

Ox 1000 A3.

Shrink the pore to 3 –5 nm by TEM

3-5 nm

Shrink the pore to 3 –5 nm by TEM

3-5 nm5.

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23 nm (1,000,000 X)

41 x 26 nm (300,000 X)

Pore shrinking and shape changing (After Thermal Oxidation, Oxide Thickness = 50 nm)

4.2 nm x 4.6 nm1,000,000 X)

19 nm (1,000,000 X)

11 nm (1,000,000 X)

7 nm (1,000,000 X)

Slopes in the plot are the shrinkage rates. Different initial pore size had

different shrinkage rates.

Pore size vs TEM shrinking time

y = -0.6034x + 48.357R

2

= 0.9638Shrinkage Rate= 0.6 nm / min

y = -0.3067x + 14.617R2 = 0.9511

Shrinkage Rate= 0.3 nm / min

0

10

20

30

40

50

60

0 10 20 30 40 50 60 70 80 90

Shrinking Time (min)

Initial diameter = 48 nmInitial diameter = 15 nmLinear (Initial diameter = 48 nm)Linear (Initial diameter = 15 nm)

168nm x 172nm 187nm x 189nm 20 min

200nm x 201nm 206 nm x 208 nm45 min 58 min

H. Chang, F. Kosari, G. Andreadakis, G. Vasmatzis, E. Basgall, A. H. King, and R. Bashir, “Towards Integrated Micro-Machined Silicon-Based Nanopores For Characterization Of DNA ”, Hilton Head MEMS conference, 2004, Hilton Head, South Carolina.

A. J. Storm, J.H. Chen, X.S. Ling, H.W. Zanderbergen and C. Dekker, “Fabrication of solid-state nanopores with single-nanometre precision”, Nature Materials, 2, 537 (2003).

9nm x 198nm 80nm x 156nmInitial pore 90 min

48nm x 57nm 3.3nm x 3.5nm250 min 334 min

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‘Nanopore Channel’ Sensors for Characterization of Single Molecule dsDNA

A

Electrode

50nm Silicon

100nm SiO2

100nm SiO2

~50-60nm

Not drawn to scale

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

H. Chang, et al., "DNA Mediated Fluctuations in Ionic Current through Silicon Oxide Nano-Channels", Nano Letters, vol. 4, No. 8, 1551-1556, 2004.

§ 200bp DNA was used. Concentration of 0.3 mg/ml.

§ Buffer solution : 0.1 M KCl, 2 mMTris (pH 8.5)

§ Ag/AgCl electrodes were utilized.§ Bias : 200 mV.§ Time sampling interval : 100 us

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Explanation of Current Pulses

K

- - - - - - - - - - -

---

-- -

--

---

--

- -

- - - - - - - - - - -

- - - - - - - - - - ---

---

---

--

---

--

- - - - - - - - - - -

I+

int

I K+ int K+

Cl -K+

Cl -

--

---

---

---

--------

-

--

---

---

---

--------

-

+ 200mV

I K+ bulk

I Cl- bu

lk

K

- - - - - - - - - - -

--

---

--

---

---

--

- - - - - - - - - - -

- - - - - - - - - - ---

---

---

--

-- -

--

- - - - - - - - - - -

I+

int

I K+ int K+

Cl -K+

Cl -

--

---

---

---

-----

---

-

--

---

---

---

-----

---

-

+ 200mV

K

- - - - - - - - - - -

---

--

----

--

---

-

- - - - - - - - - - -

- - - - - - - - - - ---

--

---

--

---

---

- - - - - - - - - - -

I+

int

I K+ int K+

Cl -K+

Cl -

--

---

---

---

-----

---

-

--

---

---

---

-----

---

-

+ 200mV

K

- - - - - - - - - - -

--

---

---

--

--

---

- - - - - - - - - - -

- - - - - - - - - - ----

---

--

---

--

--

- - - - - - - - - - -

I K+bu

lk

I Cl- bu

lk

I+

int

I K+ int K+

Cl-K+

Cl-

+ 200mV

--

---

---

---

-----

---

-

--

---

---

---

-----

---

-

DNA

DNA induces extra potassium ions when passing through the nano-channel. The interface current of K ions thus increases. At the same time bulk currents

decrease because of DNA blocking.

15Stokes, Griffen, Vo-Dinh, Fresenius J Anal Chem, 369,:295-301, 2001

Integrated Optical Detection

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Optical Detection in Biochips

1. Fluorescence: Markers that emit light at specific wavelengths and enhancement, or reduction (as in Fluorescence Resonance Energy Transfer) in optical signal can indicate a binding reaction

2. Chemiluminescence: Generation of light by the release of energy as a result of a chemical reaction. - Light emission from a living organism is termed bioluminescence (sometimes called biological fluorescence),- light emission which take place by passage of electrical current is designated electrochemiluminescence.

Capture probes

TargetProbes

Fluorescencedetection

DNA detection on chip surfaces

Capture probes

Fluorescencedetection

Protein detection on chip surfaces

TargetProbes

Capture probes

Cell detection on chip surfaces

Capture probes

TargetProbes

Fluorescencedetection

DNA detection on chip surfaces

Capture probes

Fluorescencedetection

Protein detection on chip surfaces

TargetProbes

Capture probes

Cell detection on chip surfaces

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DNA Hybridization in Microarrays

• Basis for detection of unknown nucleotides• Example: Bio-chips for identification of DNA

– Hybridization of an unknown, flourescently tagged strand with amany known strands - reaction will determine the sequence of the unknown (or vice versa)

– Strands can be lithographically (Affymetrix) or electronically (nanogen) defined at a specific location

S1 S2 S3

S4 S5 S6

S7 S8 S9

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Capture probes

+++

(d)

Metal Contact

Capture probes

Target probe (w. fluo. Label)

Attachment layerMetal Contact

Capture probes

Target probe (w. fluo. Label)

Attachment layer (c)

Capture probes

- - -

(e)

Metal Contact

Capture probes

Attachment layer

+++

(a)

Metal Contact

Capture probes

Attachment layer

+++

(b)

Electronic Placement of DNA Probes

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DNA Biochips (Nanogen)

Technology Features:• Biochips for DNA detection, antigen-antibody, enzyme-substrate, cell-

receptor and cell separation techniques. • Takes advantage of charges on biological molecules.• Small sequences of DNA capture probes to be electronically placed at,

or "addressed" to, specific sites on the microchip.

www.nanogen.com

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Technology Features

Hybridization.• A test sample can be analyzed for the

presence of target DNA molecules by determining which of the DNA capture probes on the array bind, or hybridize, with complementary DNA in the test sample.

• Fluorescence output

www.nanogen.com

21Fodor, et al. 1991, www.affymetrix.com

Light Directed DNA Synthesis on a chip (Affymetrix)

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Light Directed DNA Synthesis on a chip (Affymetrix)

Fodor, et al. 1991, www.affymetrix.com

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• Fluorescence detection• Ultimately will limit size of pixel in array

Applications:Polynucleotide arrayHIV resequencingmRNA expression monitoring

Light Directed DNA Synthesis on a chip (Affymetrix)

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Protein Arrays

G. MacBeath, and S.L. Schreiber, Printing proteins as microarrays for high-throughput function determination, Science, 289, 1760, 2000.

• Protein-Protein Interactions• Protein small molecule interactions• Derivatized substrates – glass, plastics• High Throughput screening of chemical

compounds

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Note: Sensor Arrays

• Any of the individual sensors described earlier can be used in an array format to make micro/nano sensor arrays.

• The sensors in the array need addressing• Each sensor can be functionalized with different

bio-receptor molecule to detect different entities• Examples, cantilever array, electrochemical

detection in electrode arrays, cellular arrays for chemical detection, etc.

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Lab-on-a-Chip/Integrated Devices• Single chip device for DNA

electrophoresis• Sample loading and

metering• PCR on a chip (faster

temperature cycling due to reduced thermal mass)

• Gel electrophoresis on chip

Burns, et al. 1998, Science, v 282, n 5388, Oct 16, 1998, p 484-487

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• Micro-fluidic devices on a CD type platform using centrifugal and capillary forces for liquid transport

• Cheap plastic CDs• Optical detection

systems

CD Format Biochips

Madou et al., 2001, Biomedical MicroDevices, v 3, n 3, 2001, p 245-54

28McClain, et al. Analytical Chemistry, v 75, n 21, Nov 1, 2003, p 5646-5655

• Plastic biochips using hydrodynamic transport of cells

• Electric field mediated lysing• Fluorescence detection (off-

chip detectors)• Analysis time of about 10

cells/minute

Cellular Analysis on Chip

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Polymer µSensor and Actuator

Process flow for the preparation of a hydrogel valve.

Hydrogel valve designs (2D and 3D)A biomimetic valve based on

bistrip hydrogel.D.J. Beebe et al., Proc. Natl. Acad. Sci. U.S.A. 97, 13488 (2000).

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Design of the 96-channel CAE microplate and radial scanner. Mask pattern used to form the 96 straight channel

radial microplate on a 150-mm diameter wafer.

DNA Capillary Electrophoresis

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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 ofMicroorganisms and Cells

SelectiveCapture

Ab-based

Capture

“Lab on a Chip” for Enabled by BioMEMS and

Bionanotechnology

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Micro-fluidic Polymer Devices for Culture Bacteria and Spores

• Growth of bacteria inside a micro-fluidic polymer chip

• Rapid detection and reduced time to result

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

Silicon Base, 3 PDMS layers, Top I/O port

1.0E+03

1.0E+04

1.0E+05

1.0E+06

1.0E+07

0 5 10 15 20 25Time (hours)

Impe

danc

e M

agni

tude

[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 25Time (hours)

Impe

danc

e M

agni

tude

[W]

10:1, saturated10:1, unsaturated

2.5:1, unsaturated

2.5:1, saturated

Woo-Jin Chang, Demir Akin, Miroslav Sedlek, Michael Ladisch, Rashid Bashir, , “Hybrid Poly(dimethylsiloxane) (PDMS)/Silicon Biochips For Bacterial Culture Applications”, Biomedical Microdevices 5:4, 281-290, 2003,

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Future Directions

• Integrated device for analysis of single cells – applications and fundamental science

• Building cell by cell/tissue engineering using micro and nano fabrication techniques

• Integrated diagnostics and therapeutics (drug delivery)

• Tools for genetic manipulation of microorganisms and viruses – synthetic biology

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AcknowledgementsResearch Scientists/Post-docs:• Dr. Demir Akin• Dr. Dallas Morisette• Dr. Rafael Gomez

Graduate Students:• Sangwoo Lee• Haibo Li• Amit Gupta• Hung Chang• Yi-Shao Liu• Samir Iqbal• Oguz Elibol• Angelica Davilia• Kidong Park

Industries:• BioVitesse, Inc. Co-Founder

Faculty Collaborators– Prof. D. Bergstrom (Med Chem)– Prof. A. Bhunia (Food Science)– Prof. M. Ladisch (Ag& Bio Engr)

Funding Agencies• US Department of Agriculture (Food

Safety Engineering Center)• NASA Institute on Nano-electronics and

Computing• NSF, NSF Career Award• National Institute of Health• DARPA Nanotechnology Research• Discovery Park at Purdue University

Special Thanks– Prof. S. Broyles (BioChem)– Profs. D. Datta, D. Janes (ECE, NASA

INAC), J. Cooper (BNC)

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