Introduction to Bio-Chip, Biosensors,

BioMEMS
R. Bashir
Laboratory of Integrated Biomedical Micro/Nanotechnology and
Applications (LIBNA),
School of Electrical and Computer Engineering,
Department of Biomedical Engineering,
Purdue University, West Lafayette, Indiana
http://www.ece.purdue.edu/~bashir

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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

Cells Bacteria Viruses Proteins DNA Molecules

2
 BioMEMS and Bionanotechnology
Apply micro/nano-technology to develop novel devices and
systems that have a biomedical impact or are bio-inspired

Micro/Nanotechnology
and Systems

Biology &
Biomedicine

Novel Solutions for Novel Solutions for

Frontiers in Medicine Frontiers in Materials
and Biology and Information
3
Processing
 On Size and Scale !

100µm Top-down

MicroElectronics
Plant and Animal Cells

& MEMS
10µm MEMS
Molecular Micro-fluidics
Most Bacteria Devices
& Molecule-
Feature Size

1µm Memory MEMS/ Specific

NEMS Sensors
2-D CMOS platform

100nm
Virus Min Feature
of MOS-T
(in 2004)
10nm

functional
Nanoscale
Integrated

elements
Proteins
One Helical Turn of DNA BioChips
1nm Gate Insulator (Macro, Micro,
for 100nm Nano)
MOS-T
0.1nm Atoms

Bottoms-Up 4
 Definitions

• Biosensors are ‘analytical devices that combine a

biologically sensitive element with a physical or chemical
transducer to selectively and quantitatively detect the
presence of specific compounds in a given external
environment’ [Vo-Dinh and Cullum, 2000].

• Biochips can be defined as ‘microelectronic-inspired

devices that are used for delivery, processing, analysis,
or detection of biological molecules and species’ [Bashir,
2004]. These devices are used to detect cells,
microorganisms, viruses, proteins, DNA and related
nucleic acids, and small molecules of biochemical
importance and interest.

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 Overview of Biosensor System

Sample Processing/ Detection/ Data Analysis/

• Water Separation ID Results
• Food
• Air
• Body
Fluids

6
 Introduction
Key Attributes of Biochips
1. Small length scale
2. Small thermal mass
3. Laminar flow, Re < 1
4. High surface-to-volume ratio

W.J. Chang, Demir Akin, Miroslav Sedlek, Michael

Ladisch, Rashid Bashir, Biomedical Microdevices,
vol. 5, no. 4, pp. 281-290, 2003. Whitesides Harvard University

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 Reasons for Miniaturization

• In general, the use of micro and nano-scale

detection technologies is justified by,
– (i) reducing the sensor element to the scale of the
target species and hence providing a higher
sensitivity Æ single entity/molecule
– (ii) reduced reagent volumes and associated costs,
– (iii) reduced time to result due to small volumes
resulting in higher effective concentrations,
– (iv) amenability of portability and miniaturization of the
entire system
– (v) point-of-care diagnostic,
– (vi) Multi-agent detection capability
– (vii) Potential for use in vitro as well as in vivo

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 Biochips for Detection
• Applications
o Medicine

o Pharmaceuticals

o Food Safety

o Homeland Security, etc.

• Integrated, Sensitive, Rapid, Cost x

Performance
• Commercialized; Nanogen,
Affymetrix, Caliper, Others….

Cells Bacteria Viruses Proteins DNA Molecules

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 Novel Tools for NanoBiology
Transcription factors: Controlled Microenvironment
Proteins that control the
in a Biochip
transcription of specific
genes
stimulus
DNA
Cell
Transcription Real-time cell
mRNA bio-chemical
communication
Translation
Proteins Electrical
or Optical
Signals

• Analysis of single cells and the study of their function in real time.
• Increase understanding of signaling pathways inside the cell.
• Basic cell functions such as differentiation, reproduction,
apoptosis, etc. and their implications on various disease states.
• Focus of the post-genomic era and systems biology

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 BioChip/BioMEMS Materials

• Silicon and microelectronic materials

• Glass, Quartz
• Polymers
– Poly (dimethylsiloxane) (PDMS)
– Poly (methyl methacrylate) (PMMA)
– Teflon, etc.
• Biological Entities
– Cells, Proteins, DNA
– Frontier of BioMEMS !

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 Introduction to Device Fabrication
• MEMS/NEMS Silicon Fabrication
– Formation of structures that could be used to form sensors and
actuators.
– Processing of electrical or non-electrical signals.
– Conventional and new semiconductor processing technology modules
are used.
– Etching, Deposition, Photolithography, Oxidation, Epitaxy, etc.
– Deep RIE, Thick Plating, etc
• Bulk and Surface Micromachining

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 MEMS Examples
From Dec 1996, Electron IC Design Probes for AFM

Bulk Micromachined Accelerometer

from Silicon Microstructures. Inc.

DMD Chip from Texas Instruments

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 MEMS Examples
Single Chip
Single Chip Microphone
Accelerometer Au back-plate
(Analog Devices)

Sensor

Etch Cavity
Chip

Si
Membrane
Deployment of
air-bag Draper Labs,
National Semiconductor, 1998 14
 Silicon BioMEMS Examples

Kumetrix Purdue Silicon BioChip IBM Zurich Research

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 BioMEMS/Biochip Fabrication

• In addition to Silicon….
• Biocompatibility, ideal for biomedical
devices
• Transparent within the visible
spectrum
• Rapid fabrication
• Photo-definable
• Chemically modifiable
• Possible choices
– PDMS - polydimethylsiloxane,
– Hydrogels – PMAA,
– Teflon
– SU-8, etc.
Lab on Chip (Caliper)

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 Alternative Fabrication Methods

• Soft Lithography
– Replication and molding
– Micro-contact printing
– Micro-molding in capillaries
– Micro-transfer molding
– Solvent assisted micro-molding
– Dip Pen Lithography
• Compression Molding
– Hot Embossing
– Injection Molding
• Inkjet Printing
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 Replication and Molding

• Master mold made from

silicon, glass, metal, SU-8
• Surface treatment of master
• Pour PDMS (mix, oligomer,
and CL agent)
• Cure (~60C, 1 hr)
• Peel off PDMS structure
• Mold can be used again
• Y. Gia, and G. M. Whitesides,
Annu. Rev. Mater. Sci. 1998,
28, 153-84

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 µ-Contact Printing
• Ink the PDMS structure with
molecules (alkylthiols,
proteins, DNA, etc.)
• Transfer the layer through
physical contact (optimize
time)
• Inking is performed via
covalent binding on substrate
• Can be performed on flat
surface or curved surface

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PDMS/Glass (Silicon) Hybrid Biochip

(a) PDMS 1st layer

Vertical channels
(e)
Silanized silicon mold

(b) Teflon tubing

(f)
Bonding on
top of
(c) PDMS 2nd layer silicon chip

(g)
Connection
of tubings
(d) Horizontal channel for the flow
of liquid

Opening sealed with PDMS

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 Silicon Base, 3 PDMS layers, Glass Base, 3 PDMS layers,
Top I/O port Top I/O port, Valves
Input
Reservoir
in 3rd Layer 2nd Layer
of PDMS of PDMS

1st Layer
of PDMS

Chip
Underneath

Input
Reservoir
in 3rd Layer Bonding Output
of PDMS with PDMS Tube 2nd Layer
of PDMS
Air channels

1st Layer
of PDMS
Metal

Oxide
Silicon

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 Dip Pen Lithography
• AFM Tip used to ‘write’
molecules
• Being commercialized by
Nanoink, Inc.
• SAMs, DNA, Proteins, etc.
• Serial (need array of cantilevers
for parallel writing)
• Continuous source of
molecules – microfluidics !

Lee, K.B.; Park, S.J.; Mirkin , C.A. ; Smith, J.C.; Mrksich, M.

Protein nanoarrays generated by dip-pen nanolithography 22
Science 2002 , 295, 1702-1705.
 Compression Molding
Hot Embossing Precision Injection
Molding
Mold
Heat
Pressure

Polymer
(thermoplastic
material) Features down to 0.1um
deep and 0.6um wide (for
CD-R)

Substrate

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 Nano-Imprint Lithography
Imprint mold with 10nm diameter
10nm holes imprinted in
diameter pillars PMMA

Substrate

Substrate

10nm
diameter
• Nano-scale extension of hot metal dots
embossing
• Need a nano-scale master mold
• Added to ITRS Roadmap

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Steve Chou, Princeton U.
 Key Topics

• Biochips/Biosensors and Device Fabrication

• Cells, DNA, Proteins
• Micro-fluidics
• Biochip Sensors & Detection Methods
• Micro-arrays
• Lab-on-a-chip Devices

Cells Bacteria Viruses Proteins DNA Molecules

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 Cells – Brief Overview
• Genetic information is
contained in chromatin (a
diffused mass which
distinguishes to a
chromosome when cell is
ready to divide)
• Humans have 46
chromosomes in each cell
(except in reproductive
cells)
• Chromosomes are long,
uninterrupted, packed,
super-coiled linear polymer
strands of DNA
(deoxyribonucleic acid) - 6
cm long when extended
• In humans, each
chromosome is 50-400 x
106 units long 26
From: Biology, 4th Edition by Campbell
 Cells – Brief Overview
Transcription factors: Surface Proteins
Proteins that control the (could be specific to cells)
transcription of specific genes

Y
DNA Cell
Transcription
mRNA
Translation
Proteins

Y
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 DNA to Proteins
• Transcription
– double stranded DNA is
converted to a single
stranded mRNA
– RNA polymerase
synthesizes the mRNA

• Translation
– Ribosomes ‘translate’ the
sequence of bases in the
mRNA to proteins.
– These proteins than perform
various functions inside and
outside the cell

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 Decreasing complexity Chromosomes Æ DNA

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 Structure of DNA

• DNA is composed of;

– a phosphate back-bone
where each phosphate
radical has a negative
charge
– a Deoxyribose (D in DNA)
sugar
– 4 types of bases or
nucleotides. These are
adenine (A), thymine (T),
cytosine (C), Guanine (G)
• A binds to T and G
binds to C -
complementary base
pairs

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 Purines Pyrimidines
(A, G) (T, C)
2 rings 1 ring
Structure of DNA

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 DNA Hybridization

• When DNA is heated to a temperature (~>90°C) or exposed to pH

> ~12, the complementary strands dissociates - DNA denaturation
• Process is reversible (exposure to a melting temperature Tm >
65°C) and 2 complementary ssDNA will hybridize to each other
and join to form dsDNA
• Hybridization can happen between any two complementary single
stranded molecules (DNA/DNA, DNA/RNA, RNA/RNA)
• Can provide a very sensitive means to detect specific nucleotide
sequences
• Factors affecting hybridizaton : temperature, Salt and buffer
concentration, G & C content - Tm can be calculated
• Rate of hybridization is proportional to concentration of target and
probe and limited by the lower concentration material

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DNA Hybridization

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 DNA Hybridization

Stringency
T A
A C
Reduced C G
T
G
Stringency G A
C
T
G C A
Hybridization C
A
C

T A
A C
T
C G
G
Stringent T A
C
T
A
Hybridization G G
T
G
C

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 PCR - Polymerase Chain Reaction

• Technique to amplify (make multiple copies) of

known DNA molecules - invented in 1985
• Use enzyme called DNA polymerase and primers
(short ssDNA strands)
• Billions of copies can be made within hours in
laboratory
• Very useful in research, diagnosis, forensics, etc
where large samples are required from very small
concentrations.

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 PCR Sequence

• Primers are short strands of

nucleotides which are
complementary to specific regions
of the target DNA to the amplified -
hence the ‘end’ sequence of short
1. Denature 1. Denature regions of the target DNA to be
90C
3. Extend
90C
3. Extend copied is needed
70C 70C • DNA Polymerase is an enzyme
Temperature

55C 55C which takes nucleotides from the

2. Anneal 2. Anneal
ambient solution and starts to
RT
construct the complementary
sequence
• An adequate supply of
15-30 15-30 30-60 Time nucleotides are needed (dNTPs -
sec sec sec deoxyribonucleose triphosphates -
dATP, dCTP, dGTP, dTTP)
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 Protein Structure

NH2 NH2
R C H
R1 C H
COOH Peptide Bond

R2 C H

Peptide Bond

R3 C H

Peptide Bond

R4 C H

COOH

http://www.umass.edu/microbio/rasmol/rotating.htm
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http://www.umass.edu/microbio/chime/antibody/
 Protein Structure
•alanine - ala - A
• There are 20 different amino •arginine - arg - R
acids that can make an infinite •asparagine - asn - N
•aspartic acid - asp - D
number of proteins. •cysteine - cys - C
•glutamine - gln - Q
• 3 bases within the mRNA are •glutamic acid - glu - E
called a ‘codon’. •glycine - gly - G
•histidine - his - H
• 4 different bases in combination •isoleucine - ile - I
of 3 results in 64 possible •leucine - leu - L
•lysine - lys - K
codons. •methionine - met - M
•phenylalanine - phe - F
• 3 of these are ‘stop codons’ •proline - pro - P
•serine - ser - S
• 61 specify the 20 amino acids - •threonine - thr - T
hence there is degeneracy •tryptophan - trp - W
•tyrosine - tyr - Y
•valine - val - V
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 DN A Base M -R N A
A m in o A c id
T rip le ts Codons
CG A, CG G, GCU, GCC,
a la n in e
CGT, CGC GC A, G CG
G C A, G C G , G C T, CGU, CGC,
a rg in in e GCC CG A, C G G
TCT, TCC AG A, AGG
a s p a ra g in e TTA, TTG AAU, AAC
a s p a rta te CTA, CTG G AU, G AC
c ys te in e AC A, AC G UG A, U G C
g lu ta m a te CTT, CTC G AA, G AG
g lu ta m in e GTT, GTC CAA, CAG
CCA, CCG , CCT, GGU, GGC, DNA Computer
g ly c in e
CCC GG A, GG G
h is tid in e GTA, GTG C AU , C AC Chromosome Floppy Disk
AUU, AUC,
is o le u c in e TAA, TAG , TAT
AU A
Gene File
AAT, AAC, G AA, UUA, UUG ,
le u c in e G AG CUU, CUC
G AT, G AC CU A, CU G Codon (3 bases) Byte (8 bit character)
lys in e TTT, TTC AAA, AAG
m e th io n in e TAC AUG
Base (A,T,C or G) Bit (0 or 1)
p h e n yla la n i
AAA, AAG UUU, UUC
ne
GG A, GGG, CCU, CCC, Mutation Corrupted File
p ro lin e
G G T, G GC CC A, CC G
AG A, AG G , AG T, UCU, UCC,
s e rin e AG C UC A, UC G
TC A, TC G AGU, AGC
UAA, U AG,
s to p ATG , ATT, ACT
UGA
TG A, TG G , TGT, ACU, ACC,
th re o n in e
TGC AC A, ACG
tr yp to p h a n ACC UGG
tyro s in e ATA, ATG U AU , U AC
C AA, C AG , C AT, GUU, GUC, 39
va lin e
C AC GU A, G U G http://waynesword.palomar.edu/codons.htm
 Summary
• Hereditary information is encoded in the chemical language
of DNA and reproduced in the cells of all living organisms.
• DNA is composed of a string of four basic nucleotides
referred to as Adenine, Guanine, Cytosine, and Thymine.
• In all living cells, double-stranded DNA undergoes the
process of ‘transcription’ to form single-stranded mRNA
(messenger RNA).
• The mRNA is composed of a string of four basic
nucleotides (Adenine, Guanine, Cytosine, and Uracil).
• mRNA’s undergo the process of ‘translation’ by the
ribosomes to form various proteins which then perform
and enable the critical functions of life.

• DNA - deoxyribonucleic acid (ACGT)

• RNA - ribonucleic acid (ACGU)
• Bases - nucleotides, AGTCU
• Proteins made of 20 amino acids
• RNA polymerase synthesizes the mRNA
• Ribosomes synthesize the proteins
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 Summary
• The nucleotide sequence of DNA and its expression in various cells
is of utmost importance to life scientists because every disease
state or biological function could be traced back to a single or a
group of genes (DNA sequences).
• Determination of signaling pathways of proteins is vital to
understanding the functions of cells
• Information in DNA is static, transcription and translation processes
are dynamic
• Genomics and proteomics have wide applications in biotechnology,
medicine, agriculture, biology, etc.

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 Bio-link 1: DNA
• A DNA strand is specific to its complement
⇒ Use DNA as an “address” label and
attachment system to assemble objects G C

• DNA can be attached to gold-coated objects Sugar

T A

via thiol (SH) Phosphate

– SH forms metal thiolate bond A T

G C

C G

Au Nanoclusters

1 helical turn
3.4nm
G C

C G

A T

A
T A

C. A. Mirkin, R. L. Letsinger, R. C. Mucic, and J. J. Storoff, “A DNA-based Method for Rationally Assembling
Nanoparticles into Macroscopic Materials”, Nature, Vol. 382, 15th August, 1996.
A. P. Alivisatos, K. P. Johnsson, X. Peng, T. E. Wilson, C. J. Loweth, M. P. Bruchez, and P. G. Schultz, 42
“Organization of Nanocrystal Molecules Using DNA”, Nature, Vol. 382, 15th August, 1996.
 Bio-link 2: Protein Complex
Specific interactions of antigen-antibody
• Antigen/Antibodies
Antigen
– Complicated folded structures
Antigenic
– Binding through hydrophobic, H determinant
bonds, ionic, van der Waals Antigen-
binding sites Antibody

• Ligand/Receptors Structure of one

– Avidin/Biotin sub-unit of Avidin
• Commonly used in assays
• Strong affinity (Ka=1015 M-1)

• Attachment to surfaces is more

challenging
– e.g. BSA/avidin complex
– Avidin maintains its activity when
adsorbed on oxide through BSA
– Covalent linkage on oxide through Biotinylated
Silanes molecule
http://www.rcsb.org/pdb/
http://step.sdsc.edu/projects95/Protein.lesson/avidin-biotin.html Avidin
R. Bashir, R. Gomez et al. , “Adsorption of Avidin on Micro- BSA 43
Fabricated Surfaces for Protein Biochip Applications”, Biotechnology
and Bioengineering, Volume 73, Issue 4, May 2001, pp. 324-328. Oxide surface
 Basis for Genomic Detection
Avidin Control with
coated no capture
PS beads

Biotin+
target

Capture
probes

Captured Au
nano-particles
• Thiolated DNA 1
• DNA 2 + Biotin
• Avidin coated PS beads
Æ bead capture on the
Au pads
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 DNA Capture Probes on Au Surface
Controls:
Control Sample 1) Non-thiolated attachment w/ hybridization
2) Thiolated Attachment w/ non-
8µm Au complimentary hybridization
dots Avidin coated PS beads
Æ No bead capture
Zoom Avidin
in coated
PS beads

Biotin+
8µm Au target
dots with
beads Capture
probes

Thiolated attachment
Complimentary hybridization w/ biotin
Avidin coated PS beads
Zoom
in
Æ bead capture on patterned Au
Copyright 2004, R.Bashir
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