Bio-microelectrical mechanical
systems (BioMEMS) and
Nanobiotechnology
Dr. Shalini Prasad
Electrical and Computer Engineering
Biomedical Microdevices and
Nanotechnology Laboratory
sprasad@pdx.edu
http://www.ece.pdx.edu/~prasads
ECE 510
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Course Outline
1. Introduction to Bio-MEMS and Nanobiotechnology
2. Silicon Microfabrication
3. “Soft” Fabrication Techniques
4.Polymer Materials and Microfluidics
5. Sensor Principles
6.Detection and Measurement Methods
7. Drug delivery systems
8. Micro-Total Analysis Systems (μTAS)/ Emerging Applications
9. Nanotechnology Applications
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Detection and Measurement
Methods
• Choosing a detection and measurement system
for a specific application may depend on the
preference for a labeled versus a label-free
methodology
• Some of the measurement systems are integral
to the detection scheme such as confocal laser
microscopy
• Various measurement systems are also
available for characterization of Si, glass and
polymer materials post fabrication and/or
surface modification
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Detection and Measurement
Methods
• Profilometry, is an example of a surface
measurement system
• Most techniques are used for either
detection or characterization
• All need to be miniaturized or adapted for
LOC and μTAS systems
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Detection Methods
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Electrochemical Detection (EC)
• EC liquid analysis is associated with the
measurement of electrical quantities such
as potential, current and charge in order to
gain information about the composition of
the solution and the reaction kinetics of its
components
• EC is less expensive than fluorescent
techniques
• EC detection is suitable for LOC devices
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Electrochemical Detection (EC)
• Emerging technologies with EC detection
incorporate micro fabrication of electrodes
within the device as opposed to external
electrodes
• Capillary Electrophoresis devices were
among the first to incorporate micro
fabrication of integrated electrodes
• Example- Baldwin’s work in Catechol
detection
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Electrochemical Detection
Schemes
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LOC device for Catechol Detection
• Both CE channels and all the CE/EC electrodes
were incorporated directly on to the glass
substrates via traditional micro fabrication
techniques- photolithographic patterning, wet
chemical etching, DC sputtering and thermal
wafer bonding
• Critical electrode characteristics, including size,
shape, and positions were fabricated into the
chip eliminating the need for external
electrodes.
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LOC device for Catechol Detection
• There are four CE reservoirs- sample,
waste, buffer and detection
• These are formed by drilling through the
upper glass substrate
• The corresponding Pt CE electrodes were
patterned on to the bottom plates
• Channels are 20-50 μm deep, 50-100 μm
wide and 1-2 cm long.
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LOC device for Catechol Detection
• To achieve the 250 V/cm electric fields
required for short-length CE tube, Baldwin
et al used external power supply delivering
250 V and 2500 V DC for the injection and
separation channels respectively.
• EC detection is carried out
amperometrically with a 3 electrode
potentiostat constructed from Op-amps
and passive elements
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LOC device for Catechol Detection
• The CE electrodes varying in thickness
from 0.1-0.5 μm, 500 μm-1 mm in width
and 1 cm in length.
• Pt deposits placed in each of the
reservoirs, located as far as possible from
the CE channels
• This is done to minimize the possibility that
any form of electrolysis may disrupt flow in
channel
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LOC device for Catechol Detection
• EC electrodes consist of Pt electrodes
that are 0.1-0.5 μm thick and are placed
only in the detection reservoir
• The working and reference electrodes are
20-50 μm wide semicircular Pt fingers
located at various positions on the chip
• Catechols were separated at a detection
sensitivity of μg/ml
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Integrated CE and EC
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Chemiluminescence
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Chemiluminescence
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Examples
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Chemiluminescence Detection
• Unlike, fluorescence where an excitation source
is needed and non-specific radiation can be
produced, CL occurs only when the reactants
are present and noise is inherently low.
• Detection of hybridizations using enzymecatalyzed CL reactions and micro glass array
has been performed
• A flow thru chip consisting pof 3D ordered set of
micro channels and a micro array platform is
fabricated
• Analyte specific reagents or probes are
deposited on the chip in an array of spots
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Chemiluminescence Detection
• Each spot incorporates several individual
channel sand imaging is accomplished
using a CCD camera
• Rapid bacterial detection has also been
demonstrated using this technique,
bacteria is bound to a modified CL probe
and quorum sensing based bioluminiscene
is achieved
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Chemiluminescence Detection
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Fluorescence
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Fluorescence
• When illuminated at different wavelengths,
some molecules emit light of a different
color than the excitation wavelength.
• The molecules absorb the higher energy
wavelength, lose some energy in the form
of lower energy photons.
• Also known as red shift
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Fluorescent Microscopy
• It is shown schematically in the next viewgraph.
• Excitation light is reflected off a dichoric mirror,
through the objective lens on to t he sample
• Dichromatic mirrors reflect light shorter than a
certain wavelength and pas light longer than
the wavelength
• Fluorescence from the sample is collected by
the objective lens and being of longer
wavelength ( lower energy), passes back
through the dichoric mirror to a detector
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Fluorescent Microscopy
• Epifluorescence involves illuminating the
specimen from above while in transfluorescence
the excitation light comes from below the sample
• LOC laser devices allow on-chip excitation
• Disposable integrated micro fluidic device with
self aligned planar micro lenses, LED’s as
excitation sources and photodiodes as detectors
has been demonstrated ( Seo and Lee, 2004)
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Fluorescent Microscopy
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Confocal Microscopy
• Confocal scanning microscopy has
significant utility for high density detection
such as in DNA micro arrays
• In, regular fluorescence, the entire sample
is illuminated by the excitation light
causing the entire sample to fluoresce,
therby creating a blurred image
• The addition of a pinhole filters out light
that is out-of-plane with the desired light
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Confocal Microscopy
• The object lens forms an image at the pinhole
surface and the sample and the pinhole planes
are called conjugate planes.
• The pinhole is conjugate to the focal point of the
lens thus the term confocal microscopy
• As, in fluorescence microscopy light from the
laser-higher wavelength light- is reflected by the
dichoric mirror through the objective lens on to
the sample
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Confocal Microscopy
• The fluorescence-lower wavelength-is
gathered by the objective lens , passes
through the dichoric mirror and is focused
on to the pinhole surface
• A photomultiplier tube measures fluorscent
energy.
• Moving mirrors allow the entire sample to
be scanned
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Multiple Exposure
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Rotatory Confocal Fluorescence
Scanner
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Rotatory Confocal Fluorescence
Scanner
• The Berkley rotary confocal fluorescence
scanner used to interrogate radial micro capillary
array electrophoresis devices.
• Laser excitation at 488 nm is reflected by a
dichoric beamsplitter through a hollow shaft
stepper motor and deflected 1.0 cm off axis by a
rhomb prism mounted on the motor shaft.
• Laser excitation is focused and fluorescence is
collected by the microscope objective and
reflected back through the beam splitter back to
the detector
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Rotatory Confocal Fluorescence
Scanner
• Fluorescence is spectrally filtered (dichoric
beamsplitter and band pass filter), spatially
filtered ( pinhole) and four color processed
• Oosterbroek and van den Berg (2003)
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Molecular Beacons
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Molecular Beacons
• The loop contains a probe sequence that
is complementary to the target sequence
• The stem, is formed by the annealing of
complementary arm sequences that are
locate don either side of the probe
sequence
• A fluorophore is covalently linked to the
end of one arm and a quencher is linked
covalently to the end of the other arm
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Molecular Beacons
• When MB’s hybridize to the appropriate target
nuclei strands, the probe transitions from dark to
fluorescence as the conformal changes move
the fluorophore away from the non fluorescent
quencher
• This is known as fluorescence resonance
energy transfer
• When the probe encounters a target molecule it
forms a probe target hybrid that iis longer and
more stable than the stem hybrid
• Ideal for genetic tests due to single nucleotide
sensitivity
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Molecular Beacons Applications
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Aptamers
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Example: Thrombin Aptamer
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Characterization
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Interferometry
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Interference
• Interferometers utilize the physical phenomenon
of interference patterns
• Interference occurs with the superposition of two
or more EM waves
• For coherent visible light with similar frequency
and phase and travelling in the same direcytion
this may be observed as fringes of dark and
light, which are based on addition and
subtraction of wave components at a given
point.
• These are referred to as maxima and minima
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Diffraction
• Diffraction occurs whenever a wavefront is
obstructed such as passing through a
single slit with resulting alteration of
amplitude and phase
• Fringes or diffraction patterns are formed
as some EM radiation bends around the
obstacle unto the shadow and mutually
interfere
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Interferometry
Interference fringes by splitting a beam of monochromatic light so that one
beam strikes the foxed mirror and the other the movable mirror
The beams are reflected back to the common target producing interference
patterns
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Interferometry
• Interference based integrated optical
sensors such as Mach-Zehnder
interferometer can detect refractive index
changes as small as 10-8, this
corresponds to protein concentrations of
5.3x10-8 g/ml.
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Ellipsometry
• Ellipsometry is used to measure optical
and surface properties of materials and
thin films
• In plane polarized EM radiation all
electronic oscillations are parallel to each
other and at right angles to the magnetic
oscilklations
• Light can be plane polarized by reflection
or passing through polaroid filter
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Ellipsometry
• In an ellipsometer laser light is produced
at the source and retarders shift the
phase of one component of the incident
light .
• If a light beam illuminates a thin
film/substrate surface under oblique
incidence, reflection from the surface
changes the ellipse of polarization
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Ellipsometry
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Surface Plasmon Resonance
(SPR) Spectroscopy
• SPR measurements are made using optical
systems that control the angle and wavelength
of incident light and the degree of polarization
• This technique is commonly used to study ultra
thin films with immobilized bio molecules
• A combination of substrate, noble metal and
sample allows for the generation of surface
plasmon polaritons
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Surface Plasmon Resonance
(SPR) Spectroscopy
• These are formed along the metal dielectric
interface and are highly damped charge density
waves that oscillate at optical frequencies and
may be excited if the materials and optical
properties of the experimental systems are
properly chosen.
• SPPs are not measured directly but their
presence is inferred by monitoring the light
intensity reflected from substrate/metal interface
• The reflected light contains a deep minimum in
intensity at what is termed as resonance for the
surface plasmon.
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Surface Plasmon Resonance
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Raman Microscopy
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Raman Microscopy
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Electron Microscopy
• Electron microscopes use a focused beam of
electrons for imaging rather than light
• Scanning electron microscopes use “scanning”
beam of electrons across the sample making it
possible to study, topology, morphology,
composition and crystallographic qualities of the
materials
• Topology includes surface features, texture from
which properties such as reflectivity and
hardness may be inferred
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Electron Microscopy
• Morphology is the shape and size of the
particles making up the object from which
properties such as ductility, strength and
reactivity may be inferred
• Composition includes the elements and
compounds that compose an object and
the relative abundance of their existence
that affect properties such as melting
point, hardness and reactivity.
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Electron Microscopy
• TEM is the primary tool for observation of
crystal defects including dislocations, grain
boundaries, interphase interfaces and
other planar defects
• TEM may be coupled with x-ray detectors
and electron spectrometers allowing
quantitative analysis of all the elemnts in
the samoke with sensitivity upto single
antom levels
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Electron Microscopy
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Electron Microscopy
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Atomic Force Microscopy
• Is a form of scanned proximity probe microscope
in which measuring a local property such as
height, optical absorption, or magnetism is
performed by placing a probe near the sample
• The probe sample separations are on the order
of system resolution and imaging occurs by
raster scanning the surface of interest
• AFM measures topography with a force probe,
and operates by measuring the attractive or
repulsive force between the probe tip and the
sample
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Atomic Force Microscopy
• In contact mode a cantilever tip lightly touches
the sample while deflection of the cantilever is
measured and related to height
• Measuring cantilever deflection with the angular
deflection of the laser beam allows picometer
resolution
• In non contact mode the tip does not touch the
sample but instead measures the attractive
forces.
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Atomic Force Microscopy
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Summary
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