Biosensors and Their Applications

(BIEN 515)
• Introductions
– Name
– Background
• Review of syllabus and class objectives
– Tailor this class to your individual needs!

What is a Biosensor?
• Definitions
– A device used to measure biologically relevant information
• Oxygen electrodes, neural interfaces,
– A device using a biological component as part of the transduction
mechanism
• Antibodies
• Enzymes
• DNA, RNA
• Whole cells
• Whole organs/systems

What is a Biosensor?
• Configuration
– Can be developed from any basic sensor by adding a biological
component
– Usually incorporates a biomembrane
• Transduction
– Electrical
– Optical
– Mechancial
• Mass
• Acoustic
– Thermal
– Chemical
– Magnetic
 Strain Gages, Acceler-
ometers, and Gyroscopes
• Strain Gages
– Function and parameters
– Types
– Fabrication
• Accelerometers
– Mechanics and theory
– Strain gage type
– Capacitive
– Force balanced
– Others
– Switch arrays
– Multi-axis
• Gyroscopes
– Mechanics and theory
– Vibrating beams
– Others
 Strain Gages
• Gage factor is defined as
relative resistance change
over strain
• Types include:
– Metal foil
– Thin-film metal
– Bar semiconductor
– Diffused semiconductor
• Implantable strain gages
• Penetrating micro-strain
gage probe
 Accelerometers
• F=ma is basic concept
• Force measured by deflection or strain
• Can be related to spring constant, F=kx
• Generally displacement of proof mass is
measured relative to frame
• Dynamic system as described previously
• Strain gage type most basic
– Strain in beam measured as proof mass deflects
beam
– Lots of configurations
• Capacitive accelerometers most
commercialized
– Torsion bar with assymetric plates
• Force-balanced capacitive used in autos
– Comb of capacitors measures differential
capacitance
– Highly sensitive, typical displacement only 10
nm
– Force feedback to maintain central location of
proof mass
– Force required to maintain equilibriumgenerates
signal
 Accelerometers (cont)
• Piezoelectric accelerometers
– Generally show no DC response
• Special circuitry to create DC response
– Typically use ZnO
• Tunneling accelerometers
– Highly sensitive
– More difficult to fabricate
– Requires closed loop control
– Long term drift
• Latching accelerometers
– Lock in place if acceleration exceeded
• Switch arrays
– Array of switches sensitive to increasing levels
of acceleration
– Simple to build
– Optimizes range of accelerometer in use
• Multi-axis acclerometers
– Only one example to date
– Cross-axis sensitivity problem
– Precise alignment and low cost are advantages
• All require extensive circuitry
 Gyroscopes
• Measure rotation
• Couple energy from one vibrational
axis to another due to Coriolis effect
• Two micromachined modes: Open loop
vibration and Force-to-rebalance mode
• Vibrating prismatic beams
– Beam driven in one direction,
deflection measured in orthogonal
direction
• Tuning forks
– Large inertial mass, increased
sensitivity
– Metallic ring structure
• Dual accelerometer
• Vibrating shells
– Two-axis
– Vibration in z direction
– Output in both x and y
Calorimetric Biosensors
b Principles of Operation
b Types of Devices
b Specific Examples
b Applications
b References
Calorimetry
b Used to measure heat changes involved
with thermo-chemical processes

b In general, most biochemical reactions

release heat or involve some
thermodynamic process

b Heat generation is proportional to

substrate conversion
Calorimetric Biosensors
b Rely on biological molecules for
transduction or detection
b Enzymes (or even cells) immobilized on a
surface
b Enzymes catalyze a reaction which
generates heat
b Heat is proportional to amount of substrate
present
Calorimetric Biosensor Types

b Originally highly sophisticated equipment

which was very expensive
b Three general types of sensors classified
by how heat transfer taks place between
the reaction vessel and surroundings
• Heat conduction calorimetry
• Isoperibol calorimetry
• Isothermal calorimetry
Heat Conduction Calorimetry

b Rapid heat transfer between chamber and

isothermal heat sink surrounding
b Temperature changes are measured as
voltage output of a thermoelectric sensor
between chamber and heat sink
b Heat sink kept isothermal to keep heat
transfer coefficients constant
b Thus temperature of reaction chamber is
proportional to heat generation
Heat Conduction Designs
b Miniaturized devices are potentially
implantable
b Close contact reduces need for accurate
control of ambient temperature
Isoperibol Calorimetry

b Most common method

b Also known as isothermal jacket
calorimetry
b Nearly adiabatic (no heat transfer)
b Measure temperature change in reacting
solution
b Immediate cell environment remains at
constant temperature
Isothermal Calorimetry

b Temperature of the reaction vessel is kept

constant by heating or cooling
b An adiabatic process, i.e. no heat transfer
b Heating or cooling proportional to reaction
components
Applications
b Online industrial monitoring and control
b Measurement of biomass in a sample
b Can perform immunoassays using enzyme-
labeled substrates- both direct and
sandwich types
b Blood glucose measurements as well as
metabolites (lactate and urea)
b Heavy metal and pesticide detection
Blood and Protein Separations Using a
Micromachined Electrical Field- Flow
Fractionation System
Bruce K. Galea, Karin D. Caldwellb, A. Bruno Frazierc
a Institute
for Micromanufacturing, Department of
Biomedical Engineering, Louisiana Tech University
bCenterfor Surface Biotechnology, Department of
Chemistry, Uppsala University, Sweden
cSchool of Electrical and Computer Engineering,
Georgia Institute of Technology

HSEMB 2000
 Overview

• Introduction to EFFF
• Motivation for Miniaturization
• System Fabrication and Assembly
• Basic Separations
• Biological Separations
• Conclusion
• Acknowledgments
 What is EFFF?
• Electric field forces charged particles towards wall
• Distance from wall determined by “ζ-potential”
• Relies on laminar flow profile to perform separation
function
• Distance from wall determines velocity through
channel for particles of the same size
y
x Sample Output
Sample Input
Top
+ + + + + + + Electrode + + + + + + +
20 µm

– – – – – – – – Electrode – – – – – – – –
– Bottom
 Theory- Force Balance
Electric Velocity
• Electrical and Field Profile
dispersive forces
balance to
determine thickness Diffusion
of the particle field
• Average thickness
of particle field
determines velocity
Compressed Diffuse Channel
through channel Particle Particle Wall
Field Field
 Previous EFFF Work
• First EFFF systems used to separate proteins in
1972
• First EFFF systems with non-membrane walls
built in 1993 and used to separate colloids
• EFFF has not be the subject of intense scrutiny
with only a few groups working on it, and most
for only a short time
• Progress has been slow until recent years
Separation Abilities of EFFF
• Cells and cellular components
• Viruses and bacteria
• Macromolecules (protein, DNA)
• Colloids
– Clays
– Polymers
– Liposomes, micelles, and vesicles
• Emulsions
• Surface modified particles
 Applications of EFFF
• Virus, bacteria, and organelle separations
• Characterization of emulsions, liposomes, colloids
and other vehicles for drug administration
• Diagnostic tests and molecular separations
• Protein adsorption/ surface characteristic studies
• Environmental monitoring
• Polymer, starch, and aggregation characterization
• Research on ζ-potentials
 Theory- Resolution
∆d 3 3
Lµ Veff 1
Rs = d ∝ Resolution of 44 nm and 62 nm
8 Dw 6<v> s Particles vs Width and Applied Voltage100
Rs=4
90
Rs=3
Rs=2 80
70

Width (uM)
Rs=5 60
Rs =Resolution 50
40
d = diameter of particle 30
D = Diffusivity Rs=10 20
w = Channel thickness 10
0
L = Channel length 800 1000 1200 1400 1600 1800
Veff = Effective voltage Voltage (mV)
<v> = Average fluid velocity
µ = Electrophoretic mobility
 Motivation for Miniaturization
• Shorter analysis times
• Higher resolution separations
• Parallel separation channels
• Batch or continuous fabrication with
reduced production costs
• Reduced power consumption
• All advantages mentioned previously
 Motivation for Applying
Micromachining to EFFF
• By reducing channel height the power of EFFF
systems can be increased
• Macro-machining techniques are the limiting
factor in reducing channel heights in EFFF
systems
• Possibility of integrated electronics and detection
• Precise micromachined features reduce
instrumental plate heights
 Fabrication 1
• Anisotropic etching of
input and output ports Silicon
in 20% KOH at 65 C
with Si3N4 mask Au
Conta Detect Ti
• Deposit and pattern ct or
Ti/Au electrodes on Silicon
front of wafer
• Thick photosensitive Polyimide
polyimide or SU-8
used to define flow Silicon
channels
 Fabrication 2
• Remove Si3N4
Silicon
membrane
• Deposit and pattern Glass
Ti/Au electrode on
Au
glass substrate Ti
Glass
• Bond glass substrate
to polyimide using
biocompatible UV Silicon
curable adhesive
 Fabrication 3
Output Port Input Port

• Completed channel
looking from the top Channel

Polyimide
Detector Electrode Channel Electrode

• Cross section through

channel showing
electrodes, polyimide
Completed Cross Section
and substrates
 Results- Section Fabrication

Glass substrate
with titanium,
gold, and
platinum
electrode

Silicon substrate with input/output ports, gold electrodes and patterned

SU-8
 Results-
System Assembly

Above- Complete device with

input/output port connections

Right- Complete systems with

sample and buffer input, µ-EFFF
system, and detectors
 Basic Separation Results
• Compare off-chip
3.5
detectors with on-chip 130 nm

Detector Response
3
204 nm
• Peaks clearly defined 2.5
2
• Resolution doubles 1.5 Void Peak 44 nm
1
• Time for separation
0.5
reduced significantly 0
9.90E-04 204 nm 0 5 10 15
Time (min)
9.89E-04 1.75E-07
9.88E-04 Void Peak
Detector Current (A)

Void 1.71E-07
9.87E-04 44 nm 130 nm 261 nm
9.86E-04 130 nm 1.67E-07
9.85E-04
1.63E-07
9.84E-04
9.83E-04 1.59E-07
9.82E-04
9.81E-04 1.55E-07
0.00 50.00 100.00 150.00 200.00 0 120 240 360 480 600 720
Time (sec)
 Separation by Surface Charge
• Particles can 1.2E-05

be separated 1.0E-05
by surface
Detector Response 8.0E-06
charge as well
as size 6.0E-06 High
Low
Low High
• Particles with 4.0E-06

lower surface 2.0E-06

charge density
0.0E+00
elute earlier, 0 200 400 600
even for -2.0E-06
Time (sec)
identically
sized particles
 Particles with Attached Proteins
4.90E-05 • Particles with
Protein Attached attached
4.85E-05
proteins are
4.80E-05 also retained
Current (A)

4.75E-05 Protein A
differentially
4.70E-05 Plain • Bare particles
Bare
elute ahead of
4.65E-05
particles with
4.60E-05 protein on the
4.55E-05 surface that
0 200 400 600 800 1000 1200 increases both
Time (sec) size and surface
charge
 Blood Separations
4.75E-05
• EFFF system can be used Cells
to separate some blood Hemoglobin
components 4.70E-05

• Cells are too large for Hemoglobin

Complexes
system

Current (A)
4.65E-05
• Proteins, organelles, and
Whole
mid-sized particles retained Homogenized
Platelets
9.55E-05
4.60E-05
9.05E-05
8.55E-05
8.05E-05
Current (A)

7.55E-05
7.05E-05
6.55E-05
6.05E-05 4.55E-05
5.55E-05 Whole 0 500 1000 1500 2000 2500 3000
5.05E-05 Homogenized Time (sec)
4.55E-05
0 500 1000 1500 2000 2500 3000
Time (sec)
 Conclusion
• Theory predicts benefits for miniaturization of
EFFF systems
• Design, fabrication and characterization of a
miniaturized EFFF system was performed
• Experiment confirmed basic theory
• Basic separations demonstrated
• Separation of particles with attached proteins
• Separation of some blood components
 Acknowledgments
• National Science Foundation Graduate
Research Fellowship
• Whitaker Foundation Biobased Internship
• University of Utah Technology Innovation
Grant
 DNA
b Contains genetic material for all living
organisms
b Double stranded helix
b Made up of 4 different nucleotides- A,T,C,G
b Each nucleotide a 5 carbon sugar
b Sequences of nucleotides define proteins
b Each sequence is a “gene”
Problem

b Typical molecular analysis problems

require statistically significant quantities
and must pass detection limits on the order
of millions and billions of molecules
PCR
b Technique used to produce a large number
of copies from a target DNA sequence

b Repetitive 3 step process

• Denaturation (~95ºC)
• Annealing (~55ºC)
• Chain Extension (~ 72ºC)
Basic PCR Reagents
b Template DNA
b Complementary Primers (~20 nucleotides)
b Thermostable Polymerase Enzyme (TAQ)
b Single nucleotides (A,C,G,T)
b Buffers (pH and ionic concentrations)
PCR Applications
b Creates 2n copies (typically 30 cycles)

b Genetic analysis
b Viral diagnosis
b Start with just one sample molecule
Why Apply Micromachining?
b Small reagent costs
b Fast cycling time
• Low thermal mass
• High surface to volume ratio
b System integration
• Electrophoresis
• Point of care system
b Low cost
Design Considerations
b Biocompatibility
b Chamber volume
b Control system
b Bulk or surface micromachining
b Bonding method (if necessary)
b Move the fluid or cycle in position
b External equipment
 Mechanical Pressure Sensors
• Designs
– Absolute
– Gauge
– Sealed Gauge
– Differential
 Piezoresistive Pressure Sensors
• Piezoresistivity is a material property where bulk
resistivity is influenced by mechanical stress applied
to material
• Common piezoresistors: Si, poly Si, SiO2, ZnO
• Typical design: 4 piezoresistors in a Wheatstone
bridge on a diaphragm
• Pressure sensitivity (mV/V-bar): S = (∆R/∆P)(1/R)
 Capacitive Pressure Sensors
• Capacitive sensors convert charge into change in
capacitance
• Advantages:
– more sensitive than piezoresistive
– less temperature dependent
• Disadvantages:
– gap fabrication
– diaphragm mechanical properties
Capacitive Pressure Sensors (cont)
• Basic concept: C = ε S/d
• Sensitivity: ∆C/∆d = -ε S/d2
• Small Gaps:
– larger capacitance
– easier capacitance detection
– plates may stick together
• Large Gaps:
– small capacitance
– may require wafer bonding
 Microphones
• Convert acoustic energy into electrical energy
• High sensitivity pressure sensors
• Types:
– Capacitive
• variable gap capacitor; most common
• require DC bias
• sensitivity: 0.2 to 25 mV/Pa
• response: 10 Hz to 15 kHz
 Microphones (cont)
– Piezoresistive
• diaphragm with 4 pezoresistors in a Wheatsone
bridge
• sensitivity: ~25 µV/Pa
• response: 100 Hz to 5 kHz
– Piezoelectric
• use piezoelectric material mechanically coupled to
diaphragm
• sensitivity: 50 to 250 µV/Pa
• response: 10 Hz to 10 kHz
 DNA
• Contains code for operation of all living organisms
• Made up of 4 (5) bases in long chains
• Each nucleotide a 5 carbon sugar
• Double helix of complementary strands
• Sequences of nucleotides define “genes”
• Genes define proteins
 RNA
• Converts DNA codes into proteins
• Similar to RNA with additional oxygen
• Matches up with DNA strands (complementary)
• Three types
– Messenger (mRNA)
• Carries genetic code out of nucleus
– Ribosomal (rRNA)
• Interacts with mRNA to generate protein
– Transfer
• Transports amino acids to ribosome
• Translates mRNA to generate protein
• Series of 3 RNA bases correspond to amino acid
 Proteins
• Main functional units of all cells
• Provide structure and function
• Catalytic proteins are called enzymes
• Structure of protein is critical to function
The Cell – Golgi Body
• Basic building block of • Process and sort secretory and
biology membrane proteins
• Made up of smaller – Lysosomes
• Degrade particles and
organelles dysfunctional units
– Nucleus – Mitochondria
• Site of DNA storage • Site of ATP production (power
• RNA synthesis takes place here generation)
– Endoplasmic Reticulum • ATP powers most functions in
• Smooth cell
– Processing of phospholipids and – Plasma membrane
fats
• Lipid bilayer
• Rough
– Processing of proteins
• Gatekeeper
– Studded with ribosomes • Communication
– – Cytosol
• Contains structural proteins
1) Immobilization
a) Adsorption
i) SAM’s
b) Entrapment
c) Cross-linking
d) Covalent bonding
2) Transduction efficiency
a) Highly dependent on immobilization method
b) Loading factor
i) Usually an optimal density
c) Thin layers
i) Mass transport effects
d) Specificity of attachment
i) Random attachment leads to “blocked” areas
ii) Orientation important
e) Confined attachment
i) Allows array fabrication
3) Surface modification techniques
a) Self assembled monolayers (SAM’s)
b) Light directed synthesis
c) Photolithography
d) Micromachining
e) Ink jet deposition
f) Microscopic patterning
g) Printing
4) Selectivity and Recognition
a) Structural complements
i) Hydrogen
ii) Ionic
iii) Van der Waals
b) Phase partitioning
c) Size or charge exclusion
5) Arrays
a) Multianalyte detection and analysis
b) Amplification
c) Spatial resolution
d) Averaging
6) Dimensional considerations
a) Where is measurement being made?
b) Nano-, micro-, and ultra electrodes
c) Scaling considerations and limits
i) Macro analysis may not apply at microscale
ii) Concentrations
d) Which dimension is important
e) Controllability and repeatability
7) Calibration and Figures of Merit
a) Quantifiable Performance Measures
b) Calibration uses known concentrations or samples
i) Model formation
c) Zero-order
i) One data point
ii) Comparison to theory or empirical data
d) First order
i) Array of zero order
ii) Multiple measurements in time
e) Second order, etc
f) Increasing order allows better multicomponent analysis
g) In situ calibration
h) Figures of Merit
i) Sensitivity
(1) Slope
ii) Limit of determination
(1) Based on standard deviations and sensitivity
iii) Linear range
iv) Selectivity
(1) Signal overlap
(2) Separation schemes
(3) Interferents
(4) Methods to enhance
(a) Transduction
(b) Molecular recognition
v) Response time
(1) May depend on “normal”
(2) Depends on both instrument and signal processing
(3) Measured by 95% or time constant
(4) How is sample introduced?
(5) Equilibration
(6) Refreshing time?
vi) Accuracy
vii) Precision
(1) S/N
viii) Confidence level
(1) Errors
ix) Robustness
x) Ease of use
xi) Economics
xii) Availability
 Application Overview
• Each application has its own challenges
– Applications similar to analytical chemistry
• Design and characterization (performance) heavily dependent on
application
– Example of oxygen
– Environmental variations
• Concentrations
• Media
• Temperature
• Pressure
• Interferents
• Matrix effects
– Response time

Environmental Applications
• Toxic vapors
– Hydrocarbons
• Pollution in both air and water
– Heavy metals
– Pesticides
– Herbicides
• Explosives
• Lab chemicals
• Food preparation
• Electronic nose
– Multianalyte
• Control situations

Bio-Process Control
• Pharmaceuticals
• Food processing
• Recombinant DNA
• Microbial and cell cultures
• Control (typically online)
– pH
– Temperature
– Alcohol and sugar levels
 – Dissolved gases
– Nutrients
– Waste removal
– Product collection
• In situ measurements limited by
– Sterility
– Calibration
– Lifetime
• Contamination a big issue

Clinical Applications
• Medical diagnostics and monitoring
• Trend toward decentralized and immediate results
• Generally high priority
• 10% GNP goes to healthcare
• Difficulties
– Measurements outside of controlled environment
– Miniaturization
– Stability
– Biocompatibility
– Glucose sensors good example

Ion Selective Membranes

• Allow movement of one ion while restricting another
• Allow ion selective sensing
• Membranes derived from polymers
• Complex chemistry to derive selectivity

Acoustic Wave Devices

• Mass sensitive technique
• Quartz Crystal Microbalance (QCM)
• Usually use antibodies
• Other factors affect response

Biocatalytic Sensors
• Harness enzymatic reactions
• Require immobilized enzymes
• Important considerations
– Enzyme load
– Membrane thickness
– Alternative enzymes
• Two catalytic reactions
– One substrate, one product
– Ping Pong (Two phase)

Current and Future

• Many biosensors still not commercialized
• Mostly single analyte devices
• Some arrays in production
• Unlikely to develop perfect biosensor
• Future directions
– Improved immobilization methods
– Fine tuning of molecular selectivity
• Gene and protein engineering
• Replacement of biocomponents which are expensive, unstable, and difficult to derive
– New materials
• Designed membranes to improve characteristics
• Improve connections for molecular recognition agents
– Specific binding sites on membrane
• Understanding and improving interfaces between analyte and transducer
• Transducer improvement (carbon composites, etc)
– Multianalyte sensors
– High density arrays (especially for genomics)
– Miniaturization
– Implantation
– Integration of components
– Small volumes
• Cell interiors
• Between nerves
– High spatial resolution
– Non aqueous media
– Non invasive measurements
– Improved material characterization and equipment
– Data interpretation (neural networks)
– Microseparation systems
1) Enzymes
a) Catalyze reactions
b) Kinetics
i) Study of reaction rates
c) Groups
i) Oxidoreductases
(1) Transfer electrons: H-
ii) Transferases
(1) Transfer functional groups
iii) Hydrolases
(1) Transfer functional group to water
iv)Lyases
(1) Transfer groups to or from double bonds
v) Isomerases
(1) Transfer groups within molecules
vi)Ligases
(1) Transfer by joining groups
(2) ATP cleavage
d) Optional conversion routes
i) What is final product
ii) Where is group transferred to
2) Terminology
a) Cofactor or coenzyme
i) Bond with enzyme to allow function
ii) When bound called prosthetic group
b) Holoenzyme
i) Enzyme with bound coenzyme
c) Apoenzyme
i) Enzyme without cofactor or prosthetic group
d) Activity
i) Measure of purity and ability of enzyme
ii) Given as units of activity per milligram
iii) Unit (U) defined as:
(1) Amount to convert 1 µmole of substrate/minute
(2) Given a specific pH and temperature
3) Rational design
a) Range of enzymes
i) Some enzymes catalyze same reaction but with additional
advantages
b) End products
c) Reagents
d) Consumables
e) What is being measured
i) Product
ii) Consumption
f) Interferents
i) Can they be removed?
g) Multiple sequential reactions
4) Kinetics
a) Factors related to reaction rate
b) Plots of v vs. [S]
i) Michaelis-Menten plots
ii) Assembled using multiple experiments
iii) Identical temp, pH, and enzyme concentration
iv)Assumptions (Requirements)
(1) Soluble enzymes
(2) Optimal pH for enzyme
(3) Initial substrate still in high concentration
(4) Increase in [E] increases plateau height
5) Reactions
a) E+SàESàP+E
i) k1 is reaction rate 1
ii) k2 is reaction rate 2
iii) v = d [ P] = k 2 [ES ]
dt
iv) Vmax = k 2 [ E ]tot
v) Km is [S] when ½Vmax occurs
vi)After derivation V0 = d [ P] = k2 [ES ] = k2 [ E ]tot [ S ]
dt K m + [S ]
b) Lineweaver Burke Plot
 i) Derived from above equation
ii) Constants can be read right off of plot
iii) 1 = 1 + K m 1
V Vmax Vmax [ S ]
c) Eadie-Hofstree
i) Specific to amperometric biosensors
ii) V = Vmax − K m V
[S ]
d) More complex reactions also occur and modeling has been done
for them
6) Immobilization Effects
a) Enzymatic behavior altered
b) No general trends
c) Causes
i) Random orientation
ii) Shielding of active site
iii) Denaturation
iv)Environmental effects
v) Microenvironment effects
(1) Local pH
(a)Possibly caused by polymer for entrapment
(2) Accessibility/tortuosity
(a)Cross linking of polymer
(3) Ionic strength
(4) Polarity of membrane/medium
(5) Product accumulation
7) Enzyme Inhibition
a) Determine concentration of inhibitor
b) Competitive and noncompetitive inhibition
c) Some loss of selectivity
8) Sequenced Reactions
a) Improve detection
i) Easier to detect
b) Amplification
i) Heat generation
 c) Elimination of interferents
9) Bioaffinity sensors
a) Immunoassays
b) Kinetics of “adsorption” or binding
c) On and off rates
d) Determine “Association” constants
i) Kassoc
ii) Kdissoc
iii) Typical values 104-1011 M-1
e) Similar kinetics to enzymes
f) F is fraction of available sites bound
g) Equilibrium based
i) Take minutes to hours
 Design Considerations
• Use type:
– Disposable, single use, no reagents, no training
– Portable, hand held, multiple use with a disposable component,
minimal training
– Batch testing or sample injection measurements (Large labs)
– In situ devices
• Sterilizable
• Compatible with process
– Leaching
• Minimal calibration
• No additiional reagents
– Research devices

Transduction Modes
• Electrochemical
– Potentiometric
• ADV: Easily miniaturized, easy translation
• DA: Reference required, limited linear range, pH sensitive
– Amperometric
• ADV: Variety of analytes, easily mini, dynamic range, selectivity,
• DA: Reference required, multiple membranes can be required
– Conductimetric
• ADV: Simple, easy to fab, no ref, low frequency source
• DA: Non-selective
• Optical
– Advantages
• No reference required
• Multiple modes: intensity, phase, frequency, polarization
• Real time using evanescent waves
• Multianalyte arrays simple
• Wide range of EM spectra
– Disadvantages
• Ambient light and scattering
• Limited dynamic range
• Miniatuization affects magnitude of signal
• Limited selection of chromophores and fluorophores
• Thermal
– Advantages
• Works with all reactions
• Works with all solutions
• Great for offline measurments
• Multiple analytes easily
 – Disadvanatages
• Nonselective
• Thermal enzyme probes inefficient
• Flow system required
• Mass
– Advantages
• Good detection limit
• Good for vapors
• Easily arrayed
– Disadvantages
• Nonselective
• Wave propagation problems

Analytes
• Wide range of analytes
• Wide range of enzymes
• Generally, systems reduced to some “standard” form
• Produce easily measured products
– H2O2
– O2
– pH
– NADH
– Labeled antibodies or enzymes
– Ions

Chemical Transduction
• Ion selective sensing
– Involves transport through selective membrane
• Vary parameters and materials to get different ions
– pH electrodes (some optical)
– FET
• Dissolved gases
– Often coupled to ion sensing through reaction
–
CO2 + H2O⇔H+ + HCO3-
– Gas permeable membrane
– pH change monitored using pH electrode
• Glass membrane
– Selectivity improved by going to other product
– NO important in cell signaling

Vapors
• Artificial nose
• Important in:
 – Exhalation
– Anesthetics
– Pollutants
– Nerve gases, etc
• Requires sorption
• SAWs have been used
• Neural networks for determination
• Enzyme inhibition

Biocatalysis
• See book Pg. 99 for typical reactions
• Optical, electrical, and thermal sensing available
• Multienzyme systems
– Amplification
– Recycling
– Sensitivity
– Elimination of interferents and products
– Step beyond interferents
– Suitability of sensor

Bioligand Binding
• Antibodies
• DNA/RNA
• Lectin and carbohydrates
• May have problems with dissociation and regeneration
– Increase steps for washing, etc
– May be damaging

Other Considerations
• Trace concentrations
– Ultralow concentrations
• Less than 10-9 M
– Fluorescence
– Recycling amperometry
– ELISA
• Enzyme linked immunoassays
• Temporal Resolution
– Dependent on needs
– Typically 1 minute is OK
• Spatial Resolution
– Confined spaces
– “Near” locations
– Often require micropositioning and microscopic observation
 – Interactions between “sites”
• Cross talk
• Diffusion

Mass Transport
• Analyte must reach sensor
– Internal and external components
– Time required
• Equilibrium
• Flow rate/reaction rate
• Diffusion
– Concentration gradients
• Convection
– Stirring
• Migration
– External field
• Mass transport changes in vivo
• Partitioning and permeability
• Modeling and limits
Optical Spectroscopy for Biosensing - 12/15/99
A. Definitions
1. spectrum: an array of the components of an emission or wave, separated and arranged in the order of some varying
characteristic (wavelength, frequency, mass)
2. c = λν, E= ν, =Planck's constant=6.626×10 -34 J· s
3. absorption, absorption coefficient
4. scatter, scattering coefficient, anisotropy
B. Absorption-based spectroscopy
1. Infrared spectroscopy
a. Theory
1) All atoms in constant relative vibration
2) Frequency of incident energy = frequency of bond vibration absorption
3) IR active: vibrations resulting in net change in dipole moment
4) Ranges
a) Far IR: 50-1,000µm, 200-10cm-1
Difference, coupling
b) Mid IR: 2.5-50µm, 4,000-200cm-1
Fundamental
c) Near IR: 0.78-2.5µm, 13,000-4,000cm-1
Combinations, overtones (integral multiples), coupling
*Combination of all factors Unique IR spectrum for each compound
b. Practice
1) Dispersive methods
2) FT methods - Felgett (speed), Jacquinot (throughput), Connes (internal reference) advantages
3) Transmission vs. reflectance
c. Quantitation (Beer's law) I=I0e-εlc
1) Transmittance (T): Ratio of transmitted to incident intensity (I/I0) (0-100%)
2) Absorbance (A): log10(1/T)= log10(I0/I)
3) Attenuated total reflection - evanescent wave thick or highly absorbing samples
d. Applications
1) Bioreactor monitoring
2) Food quality inspection
3) Oximetry
4) Bilirubin measurement
MAJOR ADVANTAGES: simple to perform, high SNR (FT-IR), insensitive to scatter (at longer wavelengths), multi-component
analysis*
MAJOR DISADVANTAGES: limited pathlength*, *high water absorption, *temperature dependence, *overlapping bands,
*weak absorption at short wavelengths
Photoacoustic spectroscopy
a. Theory
1) Energy absorbed converts to heat within sample
2) thermal expansion produces pressure waves
b. Practice
1) Pulsed laser source
a) Tuned to specific wavelength
b) Scanned over range spectrum
2) Focus modulated FTIR beam
2) Sensitive pressure sensor (e.g. piezoelectric)
a) single detector vs. array
b) time-based signal
c. Quantitation
d. Applications
Strongly-absorbing samples
MAJOR ADVANTAGES: highly sensitive
MAJOR DISADVANTAGES: more complex signal processing, prone to saturation at high absorption
2. Emission spectroscopy
a. Theory
1) Blackbody radiation
2) Wien's displacement law
b. Practice
Heated sample
Emission observed by spectrometer
Emission bands occur at same frequencies as absorption bands
c. Quantitation
Similar to absorbance
d. Applications
1) Temperature measurement
2) Thermal imaging
MAJOR ADVANTAGES: good for thick samples
MAJOR DISADVANTAGES: need to heat

3. Fluorescence Spectroscopy
a. Theory
1) Photon absorption Excitation of molecule to higher energy level
2) Subsequent re-emission of photon, wavelength shifted
b. Practice
1) Only small number of molecules exhibit fluorescence
2) Use known "fluorophores" to "tag" target molecules
c. Quantitation
1) F ~ concentration, Φ, intensity
2) Quenching - loss of intensity
3) May use energy transfer to advantage (Friday)
4) Photobleaching
5) Fluorescence Lifetime (Friday)
d. Applications
1) Analyte sensing
2) Imaging
MAJOR ADVANTAGES: highly specific, extremely sensitive, simple instrumentation, relatively cheap
MAJOR DISADVANTAGES: requires use of exogenous probes, limited choice of chemistry*, *short wavelengths
 C. Scatter-based spectroscopy
1. Scattering (Rayleigh) Spectroscopy
a. Theory
1) Rayleigh Scattering occurs with the interaction of light with an atom, or anything producing a refractive index
mismatched boundary
2) Scattering strength depends upon size, shape, concentration, and relative refractive index of scatterers (theory by
Mie)
b. Practice
c. Quantitation
d. Applications
MAJOR ADVANTAGES: cheap and easy
MAJOR DISADVANTAGES: nonspecific for chemistry

2. Raman Spectroscopy
a. Theory
1) Raman Scattering occurs with the interaction of light with a molecular bond.
2) Raman Scattering causes the wavelength of the light to shift
(higher = "Stokes", lower = "anti-Stokes").
3) The intensity of the Raman Scattered light is much less than the intensity of the Rayleigh scattered light
4) The amount of the wavelength shift and intensity depends on the size, shape, and strength of the molecule.
5) Similar to infrared absorption bands, each Raman shift is a distinct "fingerprint" of the molecule.
b. Practice
1) In order to see the less powerful Raman shifted light, the Rayleigh light needs to be blocked.
2) A spectrograph is used to spread out the Raman shifted light (can do FT also)
3) A photo detector or CCD camera detects the Raman shifted light from the spectrograph.
c. Quantitation
Raman Intensity ~ concentration, laser power
d. Applications
1) O2, CO2, anesthesia, and other gases expelled from a surgical patient.
2) Emissions of gases into the air by power, oil, steel, and other companies.
3) ANY application where gases, liquids, or solids need to be identified and measured.

MAJOR ADVANTAGES: no sample prep, can get depth-resolved information, PPM detection
MAJOR DISADVANTAGES: need laser&expensive filters, weak signals, high fluorescence background, scatter dependence

D. Calibration
1. Univariate
a. Peak height
b. Area under the curve
c. Ratios
2. Multivariate Calibration
a. Classical
b. Factor-based methods
3. Wavelength selection
Optical Glucose Monitors - 12/17/99

General Advantages of Optical Biosensors

Potentially Noninvasive
No Electrical Connections
High Bandwidth - Information Density

Raman spectroscopy
Need low-scattering site - Eye

Polarimetry
Relies upon rotation of plane of polarization by "chiral" molecules
Polarized laser source, polarizers aligned at 90-degrees
àintensity increases as sample rotates polarization
Need low-scattering/low-birefringence site - Eye?
Other species are chiral, not just glucose

Near-infrared spectroscopy
Many companies working on this... “Biocontrol”
Many papers published
No convincing evidence of specific measurements in vivo
More useful in process control…monitoring cell culture growthà can measure multiple species
simultaneously

Fluorescence spectroscopy
Highly-specific (Probe Required)
Invasive - Probe chemistry at tip of optical fiber
Minimally Invasive - Implanted glucose-sensitive particles, interrogated transdermally
Glucose assay
Excited with 488nm
FITC-dextran (520nm emission) and TRITC-ConA (580nm emission)
Dextran competes with glucose for ConA binding sites à dextran displaced by glucose
Energy transfer from FITC to TRITC à relative shift in peak intensity
520nm peak increases with glucose concentration
Currently, poor reversibility with this assay (others are available)
Optics of the system relatively good
*Efficiency may be increased by using two-photon excitation
 Optical Biosensors
• Optical means “electromagnetic”
• Types of measurements
– Absorbance
– Reflectance
– Fluorescence (lifetime also)
– Fluorescence quenching
– Refractive index and refraction
– Light scattering
– Polarization
• Changes measured
– Intensity
– Frequency
– Phase shift
– Polarization
• May require source and sensor
• Spectral components important
Types and Components
• Fiber optics
• Guided waves
• Evanescent waves
• Surface plasmons
• Light emitting diodes
• Total Internal Reflection Fluorescence (TIRF)
• Interferometers and gratings
• Charge coupled devices (CCD)
• Spectroscopy
Developments
• Fiber optics for remote sensing
• Usage from evanescent waves generated during total internal
reflection
• Miniaturization
• Near field optics
• What is the simplest and easiest optical sensor?
Methods
• Capillary optical sensors
– Biomaterial immobilized in capillary
 – Fiber optics collect light output from capillary
• Reflectance interference spectroscopy
– Reflected light from thin film interferes with light reflected from other
surface
– Small changes in thickness can be measured
• Adsorbed or bound analyte
• Near IR
– Chemometric spectroscopy
– Heavily analysis dependent
Fiber Optic Devices
• Wide range of optical fibers
– Application depends on light and spectral components
– Match to source and detector
• Arrays of fibers can give multicomponent info and spatial resolution
• Cladding (IR smaller than fiber) used to reduce signal loss down fiber
• Guided waves
– Refractive index changes determineactivity
– Large angles: refraction
– Critical angle: Total internal reflectance
– Small angle: reflection
– “Cone of acceptance” or Numerical Aperture (NA)
– Modes
Evanescent Wave
• Total internal reflection generates interference
• Creates a standing wave
• Wave extends beyond the boundary of the wave guide
– Evanescent wave
• Exponentially decays from surface
– Must be close
– Less than 100-200 nm
– Ideal for chemically bound layers
• Cladding may be stripped and replaced with biomembrane, etc
Fiber Optic Design
• See page 274 of text
• Optical fiber carries light to biocomponent
– Absorbance or fluorescence is measured
• Light brought in by one fiber, collected by another
• Evanescent wave along fiber
• Planar wave guide
• Fiber bundles
• Generally very versatile
Planar Waveguides
• Similar to fiber optic devices
• Can add coupling prisms
Near Field Sensing
• Eliminates need for high energy beams
• Improved resolution
• Collection through small appertures
• Small magnitudes
• Micropositioning
 Surface Plasmon Resonance
• Works with thin films
• Most useful for Biospecific Interaction Analysis (BIA)
– Kinetics of binding
• Requires polarized light
• Only on metal surface
– Sea of electrons
– Waves on “sea” absorb light at specific angles
• Quantum mechanical detection
• Only occurs with light at an angle
– Angle depends on film thickness and refractive index
– Very sensitive
• Experimentally observed as sharp minimum in intensity measurement
SPR Applications
• Gold and silver films most often used
– About 5 nm in thickness
• Proteins or antibodies bound to surface
• Light injected from opposite surface
– Must be polarized- controlled to thousandths of a degree
• Light undergoes total internal reflection
• Evanescent wave couples to opposite layer
• Reflected light monitored
– Shifts indicate change in film thickness
Optical Sol-Gel Sensors
• Transparent gels for entrapment of biochemicals
• Allow direct spectrophotometric measurements
• Reactants diffuse into gel
• Absorbance or luminescence measurements possible
• Response time diffusion dependent
• Entrapment has little effect on biomolecules
• Limited to low molecular weight analytes
Luminescence
• Requires light generating molecules
• High energy molecules or systems
• Chemiluminescence most fundamental
• Bioluminescence is enzymatically catalyzed chemiluminescence
• Energetics
– Blue (400 nm) 70 kcal/mol
– Red (700 nm) 40 kcal/mol
 – ATP 7 kcal/mol
– ROOR- 100 kcal/mol
Chemiluminescence
• Most basic is luminol
• Generates a peroxide in presence of oxygen
• Works in any assay involving oxygen, peroxide or peroxidase
• Can be linked with fluorescence to make dual measurements
• All chemiluminescent systems are also fluorescent
• Need highly exothermic reaction
• Quantum mechanical pathway required
• Usually ring structures
• All use oxygen
Bioluminescence
• Relatively slow (3-20 s)
• Components
– Oxidant
– Luciferase or photoprotein
– Chemiluminescent substrate
• Luciferin
• Redox and electron transport
– Cofactors
• ATP or NADH
– Fluors
– Binding proteins for Protection
• Two main types
– Firefly
• Creates peroxide
• Quantum yield nearly one
– Bacteria
• Flavins as substrate
• Quantum yields 10-30 %
1) Photodetectors
a) Silicon based generally
b) Response independent of wavelength
(above a certain point)
c) Many different arrangements
d) Based on electronic bandgap in
semiconductors
e) Avalanche Photodiodes
i) Junction based system
ii) High voltages magnify signal
iii) Random process generates noise
f)
2) Charge Coupled Devices (CCD)
a) Metal – insulator – semiconductor
devices (MIS)
b) Incident light generates charge on gate
i) Induced voltage causes charge separation
c) CCD cameras are arrays of MIS devices
i) Can be 10 microns square
d) Charge collected and moved sequentially
to edge of device
e) Dyes can be applied to transistor to
detect colors
f)
3) Photomultiplier Tubes (PMT)
a) Photoemissive devices
b) Generally run at low pressures
c) Any incoming energy over work
function of cathode generates cascade
d) Noble gases in chamber can be ionize
i) Signal to noise may drop due to shot noise
e) Mulitple cathodes can be included to
create cascade (PMT)
i) No gas ionization for these systems
f) Extremely high gain (107)
g) Very low noise
i) Noise is amplified

ii)
1) Amperometric Sensors
a) Measures current flow in system
i) Spontaneously generated
(1) Microbe activity
ii)Directed by an applied voltage
(1) More typical
iii) Can be linked systems
(1) Enzymes attached to antibodies
iv) Setups
(1) Attached/ immobilized biocomponent
(2) Three types shown below
(a) Also, carbon fiber electrodes

b)
2) Methods
a) Basic materials
i) Au, Pt, C, Carbon composites, and conducting salts
(1) Material limits range of applied voltages
ii)Solvent can also limit range of applied voltage
b) Operation
i) Potential step
(1) Applied potential critical to experiment
(a) Overpotential may be required
(i) Varies with material
(ii) Highly empirical
(b) Scanning methods may be helpful to find
optimum
(2) Current measured for ms to seconds
(a) Double layer charging and electron transfer
(3) Chronoamperometry
(a) Current with time
(4) Chronocoulometry
(a) Current is integrated to get charge transfer
(5) Amperometry
(a) Sample current at specific time
ii)Mass transport governs
(1) Diffusion through membrane
(2) Alleviated by allowing electrode to go to
steady state before introducing analyte
iii)
iv) Can measure variety of species (see sensing
options)
(1) Note limitations of each sensing option
(2)

(3)
(4) Cyclic voltammetry

(5)
1) Electroanalytical Biosensors
a) Involve charge and electron transfer
i) Potential
ii) Current
iii) Conductance
iv) Impedance
b) Most common for practical devices
c) Wide variety of methods (see chart page
208)
d) Amperometry
i) Current measurement
e) Voltammetry
i) Current as a function of variable voltage
f) Chronoamperometry
i) Current as a function of time at constant
voltage
2) Electrochemistry
a) Electrochemical cell
b) Galvanic
i) Spontaneous reaction generates current or
voltage
c) Electrolytic
i) External energy source
ii) Current flows related to
oxidation/reduction levels
d) Cyclic voltammetry
i) Scan between voltages
e) Nernst potential and equation
i) O+neàR
ii) E = E + RT
o
ln
[O]
nF [R]

iii) Standard hydrogen electrode is basis

iv) Practical use, silver/silver chloride or
calomel (Hg)
(1) Typically difficult in practice,
especially in vivo
(2) Short duration
v) Microsystems avoid some of the
problem by using low currents
3) Potentiometric sensors
a) Ideal for ions and dissolved gases that
produce ions
b) No current flow (critical assumption)
c) Systems, while simple in appearance,
often quite complex
d) Polymer membrane
i) Limit access
ii) Selective to specific ions
e) Solid state
i) Must convert ions to electrons
ii) Problems with stability and
reproducibility
iii) Metal wires with applied films
iv)
4) Field Effect Transistors
a) Current from source to drain related to
gate voltage
b) Application of membranes to gate allows
selective measurements
c) Problems
i) Membrane adhesion
ii) pH sensitivity
iii) Drift
iv) Coatings can help eliminate all
v) Nonlinear
1) Amperometric Sensors (cont)
a) Oxygen species
i) Several potentials at which charge transfer occurs
ii) Some byproducts are highly toxic
iii) Addition of appropriate enzymes can limit toxicity
b) NADH
i) Involve dehydrogenases
ii) Potentially hundreds of related reactions
iii) Requires soluble coenzyme
(1) External reagent
(2) Expensive
(3) Difficult
iv) Mediators improve process
(1) Enhance electron transfer
(2) Lower required voltage
(3) Coating on electrode
c) Detection limits
i) Very low detection limits
(1) Especially with amplification
ii) Immunosensors with attached enzymes
2) Other Electronic Biosensors
a) Conductivity and Impedance
i) Inherently non-selective
ii) Require modified surfaces for selectivity
iii) No reference electrodes required
iv) Easy and inexpensive to fabricate
b) Most basic configuration is Wheatstone bridge
c) Both time and frequency domain measurements possible
d) Involve both capacitive and resistive elements
i) Magnitude and phase measurements
e) Added enzyme sensors paired with reference for differential measurement
i) Several relevant enzymes have been used
f) One example included pH sensitive hydrogel on surface of electrodes
i) Change in pH changed resistance through membrane
g) Impedance Spectroscopy
i) Impedance sweeps
ii) Similar applications to optical spectroscopy
(1) Not as “varied”
3) Bioelectric Interfaces
a) Measurement of electrical activity in the body
b) Generally very low signal levels
c) Non-selective
d) Neural Recording Arrays
i) Attempt to access multiple neurons simultaneously
ii) Attempts at cortical prostheses
iii) Used for both recording (sensor) and stimulation
e) Regeneration neural electrodes
f) Cell interfaces
g) Cultured cell systems
Microfluidic Sensors
Example University of Washington H Sensor
Methods for Fabrication
KOH etching of silicon
Other etching methods (High Aspect Ratio)
LIGA
Molding in PDMS
Interesting Ideas
Bubbles for added power
CD systems
Systems in Ceramics, Polymers, Silicon
Surface tension valves
Problems
Valves
Pumps
Seals
Mixing
Diffusion
1) Antibodies
a) Produce by body in response to antigens
b) Have specific binding domains
i) Epitope or determinant
c) Y shaped in general
i) Symmetric
d) Heavy and light chains
e) Binding site at tip of Y
i) Made up of 20-30 amino acid sequence
f) Base of Y involved in activation of
complement and other immune components
g) Polyclonal
i) Antibodies from a variety of immune cells
that bind to an antigen
ii) Usually have different epitope
h) Monoclonal antibodies
i) All produced by same cell
ii) Generated by cloning
iii) These are typically used in biosensors
2) Immunoassay
a) Based on Ag-Ab binding
b) Homogenous
i) No washing steps
ii) Free and bound antibody does not need
to be separated
iii) Usually binding causes change that
allows bound and unbound antibody to be
distinguished
c) Heterogenous
i) Require washing and separation
ii) Displacement
(1) All sites are filled with labeled
antigen
(2) Unlabeled antigen displaces labeled
antigen
iii) Competitive
(1) Labeled and unlabeled antigen added
simultaneously
(2) Amount of labeled antigen bound
gives info
iv) Sandwich
 (1) Capture antibody is immobilized
(2) Analyte is added and bound to
antibody
(3) A labeled antibody is added
generating signal
(4) Requires antigen with two
determinants
d) ELISA
i) Enzyme linked immunoassay
1) Random Homogenous Immunosensor Types
a) CEDIA
i) Clone enzyme donor immunoassay
ii) Engineered antibodies and enzymes
iii) Competitive assay
iv) Existence of analyte allows active
enzymes to form
v) Convert chromogenic substrate to dye
vi) Linear
(1) Most competitive assays are non-
linear
vii) Signal proportional to analyte present
b) ARIS
i) Apoenzyme reactivation immunosystem
(1) Competitive assay
(2) Allows enzyme activation in the
presence of antigen
c) SLFIA
i) Optical system
ii) Enzyme release fluorogenic substance in
presence of analyte
iii) If not bound to antigen, antibody blocks
formation of fluor
d) EMIT
i) Matched enzymes and antibodies
ii) Antigen activates enzyme
e) ICS
i) Ion Channel Switch
(1) Measure of changing conductance
across a membrane
(2) Competitive assay
(a) Conductance increases if analyte
present
(3) Sandwich assay
(a) Conductance decreases if analyte
present
f)
Thermal Sensors
a) Based on heat generated
b) H=Cp∆T
c) ∆T not always large
i) Requires amplification
ii) Multiple enzymes produce multiple
reactions
iii) Good thermistors and thermopiles can
detect 10-5 K
d) Specific heat can be modified using
different solvent
i) Enzyme function in other solvents not well
known
e) Thermistor
i) Resistor whos value changes with
temperature
f) Thermocouple
i) Based on Seebeck effect
ii) Dissimilar metals generate voltage
difference at interface
g) Thermopile
i) Serially linked thermocouples
Acoustic Devices
(a) TSM- Shear Thickness Mode
(b) SAW- Surface Acoustic Wave
(c) STW- Surface Transverse Wave
(d) FPW- Flexural Plate Wave
(e) SH-APM- Shear Horizontal Acoutic
Plate Mode
(f) Challenges
(i) External changes affect resonant
frequency
1) DNA Hybridization Arrays
a) Manufacturing
i) Mechanical printing
(1) Soft lithography
ii) Ink jet
iii) Photolithography
b) Applications
i) Gene expression
(1) Usually looking for RNA expression
(2) Differences between cells
(3) Differences in time
ii) Polymorphisms
(1) Change in base pairs
(2) Often looking for single base pair
change
iii) Comparative genomic hybridization
(1) Compare entire genomes
(2) Cannot detect small deletions
c) Problems
i) Sampling
(1) Homogenous cell samples
(a) Types, cell cycle
(b) RNA quantity for single cells
 (i) Requires amplification
d) Detection
i) Usually fluorescence
ii) Can use multiple tags to distinguish
samples
2) PCR
a) DNA Amplification technique
b) DNA collection
i) Cell lysis
3) LCR
a) Amplification technique and sensor in
one (if desired)
b) Similar to PCR
c) Uses DNA ligase rather than polymerase
d) Binds two strands together at the end
after binding to complementary strand
e) Sensing accomplished by using strands
with attached fluor
i) If binding not possible, fluor is lost in
washing step
1) Chromatography
a) Separation Systems
b) Usually incorporate
i) Carrier (mobile phase)
ii) Stationary phase
iii) Sample
c) Gas Chromatography
d) Liquid Chromatography
i) Ion-Exchange
ii) Normal Phase
iii) Reverse Phase
iv) Gel Permeation
v) Affinity
vi) Hydrophobic
e) Size exclusion chromatography
f)Electrophoresis
g) Field-Flow Fractionation
i) Multiple types