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OPTICAL BIOSENSORS

Week 4

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Transducer

A transducer as a device that converts an observed change (physical, chemical or

biological) into a measurable signal. In biosensors, the latter is usually an electronic signal

whose magnitude is proportional to the concentration of a specific biochemical or set of

biochemicals.

Schematic layout of a (bio)sensor.

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Schematic layout of a (bio)sensor

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Optical Transducers

Absorption spectroscopy,

Fluorescence spectroscopy,

Luminescence spectroscopy,

Internal reflection spectroscopy,

Li ht scatterin .

DNA and Protein Biochips

Surface plasmon resonance

Ellipsometry and

Fiber Optics

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Light is electromagnetic radiation of a wavelength that is visible to the human

eye (in a range from about 380 or 400 nanometres to about 760 or 780 nm).

Fundamentals

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In optics and physics, Snell's law (also known as Descartes' law, the SnellDescartes

law, and the law of refraction) is a formula used to describe the relationship between the

angles of incidence and refraction, when referring to light or other waves passing through

a boundary between two different isotropic media, such as water and glass.

Ibni- Sahl (984, AD)

Fundamentals

Refraction of light at the interface between two media of different

refractive indices, with n2 > n1. Since the velocity is lower in the second

medium (v2 < v1), the angle of refraction 2 is less than the angle ofincidence 1; that is, the ray in the higher-index medium is closer to the

normal.

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Polarized Light

The electric field vector oscillates in one direction only =>

linear polarized light

E

Fundamentals

Polarization is a property of certain types of waves that describes the orientation of their oscillations.

rec on o

propagation

Two superimposed perpendicular light waves with

equal amplitude and no phase shift

pp

ss

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Circular and elliptically polarized light

p

Two superimposed perpendicular light waves with equal

amplitude and /4 phase shift =>Circular polarized light

Fundamentals

s

p

s

Elliptically polarized light => the electric field vector

moves like a deformed coil, i.e. viewed from end-on

it describes an ellipse

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The Diffraction of Light

p

p

s

s

E E

Medium (0)

Fundamentals

1

0

sin

sin

c

cn ==

kinN~

=k = extinction coefficient

n = index of refraction

i = imaginary number ( -1 )

Medium (1)

Index of refraction(material is not absorbing; k = 0)

Complex index of refraction(material is absorbing, k 0)

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In a system with more than one interface addition of the reflected waves leads to an infinitegeometric series for the total reflected amplitude R (Total reflection coefficient)

Ambient (0)

~

0N~

# 1# 2 # 3

Fundamentals

)i(p

12

p

01

)i(p

12

p

01

p

in

p

3#out

p

2#out

p

1#outp

err1

err

E

EEE

+

+=

+++=

K

R

)i(s

12

s

01

)i(s

12

s

01

s

in

s

3#out

s

2#out

s

1#outs

err1

err

E

EEE

+

+=

+++=

K

R

== cosN

~d4shiftphase 1

Film (1)

Substrate (2)

1

2N~

d

Interference of two waves leads to a

resultant wave (light green one). The

amplitude of it depends on the phaseshift ().

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Total internal reflection, is based on measuring the intensity of a light beam reflected at an interface for

which the refractive index n1 is larger for the incident medium than the refractive index n2 of the

reflecting medium. If the light hits the interface at an angle of incidence larger than the critical angle

c definedby

Fundamentals

TOTAL INTERNAL REFLECTION

it will not be transmitted into the second medium at all and total reflection occurs. However, the

electric field of the light penetrates into the second medium and is called the evanescent field.

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The evanescent field, associated with total internal reflection, can then be used to monitor

layers at the interface. Total internal reflection is commonly used to study properties of

adsorbed layers. The evanescent field has an extension of several hundred nanometers andcan be used as an optical probe if one or more additional layer(s) are present at the

interface. If adsorption on a different material than that of the incident medium should be

Fundamentals

studied, a thin semitransparent layer can be deposited on the substrate. A very sensitivesensor approach utilizing this is the SPR technique in which a thin metal layer is deposited

on a glass prism and adsorption on the metal layer is monitored as changes in the SPR

phenomenon.

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ELLIPSOMETRY

&

ELLIPSOMETRY BASEDBIOSENSORS

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Ellipsometry is based on measurements of the change of the polarization state of a light beam

reflected from a surface. It has a high surface sensitivity, which makes it powerful for studies of

thin films . The quantities measured by an ellipsometer are the so-called ellipsometric angles and

, which are defined by the complex-valued ratio of the reflection coefficients Rp and Rs for light

polarized parallel (p) and perpendicular (s) to the plane of incidence such that

Fundamentals

pp

In principle is the amplitude ratio and the phase difference between Rp and Rs. Because these

reflection coefficients depend on the optical properties and composition of the substrate and

overlayers, on their thickness and morphology, and on surface roughness, ellipsometry is exploited

as a non-invasive probe of thin films and interfaces.

ss

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Nulling Ellipsometry

Substrate

Analyzer

Objective

Polarizer

CompensatorSample (e.g. film)

Fundamentals

= 2p + /2 (polarizer angle at minimum signal)

= a (analyzer angle at minimum signal)

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Light Source

1. The light source consists of wavelengths in the following

regions

Ultraviolet

185nm 260nm

0.4nm 0.7nm

Infrared

0.7nm 1.1m

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SWE Components and Functions

2. Polarizer - produces light in a special state of polarization at the output

3. Compensator - used to shift the phase of one component of the incident light

Depending on orientation, it transforms the ellipse of polarization

the linear polarization axis.

4. Analyzer second polarizer that detects the linearly polarized light reflected

off the sample

5. Detector

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How does Ellipsometry work?

1. Light from a light source.

2. The light is polarized by passing through a linear polarizer.

3. The light is then elliptically polarized by passing through a compensator.

4. The light hits the sample, is reflected and is linearly polarized.

5. The analyzer detects the change of polarization.

6. The detector catches the light and send it to the computer to process the data.

7. The measured data combined with computerized optical modeling gives information of the

film thickness and refractive index values of a sample.

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Single-wavelength ellipsometry employs a monochromatic light source. This is usually a laser in the

visible spectral region, for instance, a HeNe laser with a wavelength of 632.8 nm. Therefore, single-

wavelength ellipsometry is also called laser ellipsometry. The advantage of laser ellipsometry is that

laser beams can be focused on a small spot size. Furthermore, lasers have a higher power than broad

band light sources. Therefore, laser ellipsometry can be used for imaging. However, the experimental

output is restricted to one set of and values per measurement. Spectroscopic ellipsometry (SE)

employs broad band light sources, which cover a certain spectral range in the infrared, visible or

.

corresponding spectral region can be obtained, which gives access to a large number of fundamental

physical properties. Infrared spectroscopic ellipsometry (IRSE) can probe lattice vibrational (phonon)

and free charge carrier (plasmon) properties. Spectroscopic ellipsometry in the near infrared, visible

up to ultraviolet spectral region studies the refractive index in the transparency or below-band-gap

region and electronic properties, for instance, band-to-band transitions or excitons.

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Ellipsometric sensor systems of class afiinity layer (AL) are

based on affinity mechanism. In an AL sensor, a sensing layer is

deposited on a sunstrate and ideally only specific interactions

with the molecules to be detected should take place. The physical

parameter measured is the change in the effective layer thickness

often expressed as the change of surface concentration.

Fundamentals

In sensors of class matrix layer (ML), the sensing takes place inside a thin

(10-1000nm) surface layer- the matrix. This matrix maybe a porous layer

into which molecules can diffuse and interact with its internal surface. I gas

sensors capillary condensation is useful. The matrix may be rigid like in

porous silicon whereby the mechanism is pore filling resulting in a

refractive index change. The layer can be also an extended low-density

matrix to which sensing molecules are bound. Fr low density matrices both

thickness and refrative index changes may occur. Porous layers and low-

density matrices provide effective ways of increasing the surface area of

sensor.

d l

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Fundamentals

In sensors class of integrating layer (IL) the basic idea is to

monitor the change in layer thickness in situ. Note that thickness

change can be both positive (growth) and negative (erosion) and

that the sonsor is integrating in the sense of responce is

accumulated thickness change over time (Ex, corrosion and

enzymatic digestion).

In class homogenous layer (HL) ellipsometric sensor systems,

homogenous layers are used and physical/chemical influences on

the layers are monitored. Examples aere thickness and/or

refractive index changes due to temperature or pressure change.

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Schematic illustration of ellipsometric multi sensing baded on multiple beams and

samples. The light source is a laser diode and the detectors are photodiodes.

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LITERATURE

1

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1

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2

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2

2

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2

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SURFACE PLASMON RESONANCE

(SPR)

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Surface Plasmon Resonance (SPR)

What is Surface Plasmon Resonance?

Measuring of the interaction between a wave vector(induced by a light beam) and a wave vector in a metal

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SURFACE (Glass coated thin layer of Gold or Silver)

PLASMON: Oscillation of free electrons of the metal


RESONANCE: Resulted in a resonance at the metal

(gold) surface

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Surface Plasmons are charged density waves propagating

along the interface of a metal and dielectric media.


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When a light beam travels through an optically dense medium and reaches an interface of

lower optical density, it is reflected back,

In SPR monochromatic, p-polarized (parallel to the incident plane) light is focused on the

interface of the two optically dense media separated by a thin metal film,

Surface plasmon has only the electric field component normal to the surface .

S f Pl R (SPR)

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(A) In the Otto setup, the light is shone on the wall of a glass block, typically a prism, and

(A) (B)


totally reflected. A thin metal (for example gold) film is positioned close enough, that the

evanescent waves can interact with the plasma waves on the surface and excite theplasmons.

(B) In the Kretschmann configuration, the metal film is evaporated onto the glass block.

The light is again illuminating from the glass, and an evanescent wave penetrates throughthe metal film. The plasmons are excited at the outer side of the film. This configuration is

used in most practical applications.

S f Pl R (SPR)

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Although the beam is reflected, a part

of the light enters the interface of the

less dense medium to a distance of

one wave eng t.

This evanescent wave excites

molecules near the interface

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The evanescent wave of the incoming light couples with the free oscillating electrons

(plasmons) in the metal film at a specific angle of incidence, this effect reaches it

maximum when:

Kev= Ksp

This effect transfers energy from the incident light into the metal film, decreasing the

intensity of the outgoing light, which can be detected by the detector.

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When the incoming light is monochromatic and p-polarized (i.e. the electric

vector component is parallel to the plane of incidence), the free electrons of the

metal will oscillate and absorb energy at a certain angle of incident light.


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The refractive index near the sensor surface changes because

of binding of macromolecules to the surface.

As a result, the SPR angle will change according to the amountof bound macromolecules.


There is a linear relationship between the amount of boundmaterial and the shift of the SPR angle.


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Scanning mirror biosensors measure the SPR angle shift

in millidegrees as a response unit to quantify the binding

of macromolecules to the sensor surface.

The response also depends on the refractive index of the

bulk solution.


A change of 120 millidegrees represents a change in

surface protein coverage of approximately 1 ng/mm2, or in

bulk refractive index of approximately 10-3.


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( )

SUMMARY

If a molecule is adsorbed, intensity decreases and therewill be an increase in the angle (resonance unit.)

Monitoring of Biomolecular Interactions by Using SPR

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Peptide/protein protein

DNA/RNA - protein

protein - cell

receptor - cell

protein - virus/phage

carbohydrate - protein

carbohydrate - cell

liposome - protein

artificial materials - biological matter

drugs - protein

drugs - DNA/RNA.


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Applications

Affinity

Kinetics

( )

Stoichiometry

Thermodynamics

Activation energy


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A typical SPR experiment involves several discrete tasks.

Prepare ligand and analyte.

Select and insert a suitable sensor chip.

Immobilise the ligand and a control ligand to sensor surfaces.

Inject analyte and a control analyte over sensor surfaces and recordresponse.

Regenerate surfaces if necessary.

Analyse data.

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Affinity Constant

In principle the affinity constant can be measured directly by equilibrium

binding analysis, or calculated from the kon

and koff

. It involves injecting a series

of analyte concentrations and measuring the level of binding at equilibrium. The

relationshi between the bindin level and anal te concentration enables the

affinity constant to be calculated.


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Affinity Constant


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A: Injection of mAb (Antibody against TMV)

B: mAb is replaced by running buffer

Difference between B and C correspondsthe difference in refracttive index between

the mAb and running solution.

C: Indicates max. BindingD: HCl injection to remove mAbs bound to TMV


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Advantages

Allows to study interactions between molecules in real time No need for labelling

Time saving

No change of the native condition of the biomolecules

Molecules are not destroyed during monitoring

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Surface Plasmon Resonance Imaging (SPRi)

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(a) Kretschmann configuration for SPRi; a high refractive index prism is in contact with the detection cell

and couples the incident light to the surface plasmons by evanescent waves. p-polarised light is directed to

the prism, on which the biomolecular probe is tethered, and a CCD camera collects the output signal as

variations in reflectivity.

Surface Plasmon Resonance Imaging (SPRi)

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(b) Data are recorded as intensity variation of the reflected light at a fixed angle for each ROI selected. A

differential image (left) is produced in real time together with the relative sensorgrams. As example, two

sets of signals are reported here corresponding to the interaction with different chemically modified areas.

During the specific interaction with the target analyte, only the relatively specific probe will react, leading

to a local change in intensity of reflected light. This translates into black/white contrast for the image. The

unspecific receptor series (used as negative control) will give negligible or no signal. (c) Sensorgrams

corresponding to the interactions of the analyte with the spots on the surface.

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LITERATURE

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1

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1

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2

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2

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2

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2

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Fiber-optic biosensors (FOBS) use optical fibers as the transduction element, and rely

exclusively on optical transduction mechanisms for detecting target biomolecules

Fundamentals

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exclusively on optical transduction mechanisms for detecting target biomolecules.

Optical fibers consist of a cylindrical core and a surrounding cladding, both made of silica, as illustrated

in Figure. The core is generally doped with Germanium to make its refractive index. slightly higher

than the cladding refractive index, which results in light propagation by total internal reflection (TIR).

Light propagating through an optical fiber consists of two components: the guided field in the core and

the exponentially decaying evanescent field in the cladding. In a uniform-diameter fiber, the evanescent

field decays to almost zero within the cladding. Thus, light propagating in uniform-diameter cladded

fibers cannot interact with the fibers surroundings.

(A) Step-index SM fiber. Typical diameter of the core is

812m and the overall diameter is 125m. Light is

transmitted in a single mode, meaning a single path. (B)

Step-index MMfiber. Typical diameter of the core is 50

200m and the overall diameter is 125400m. Light

propagation occurs via many paths. Total internalreflection occurs when the incident angle is greater than

the critical angle.

Fundamentals

While optical fibers were originally intended for light propagation with minimal loss, it was not long

before that they found use in sensing which requires light to interact with the fibers surroundings One

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before that they found use in sensing, which requires light to interact with the fiber s surroundings. One

way to achieve this interaction is to expose the evanescent field of the transmitted light. For example, if

the cladding of a fiber is reduced or removed, the evanescent field can interact with the surroundings.

The distance to which the evanescent field extends beyond the core-cladding interface is described by

the penetration depth, which is the distance where the evanescent field decreases to 1/e of its value at the

core-cladding interface and is mathematically described as:

E(x) = E0 exp(x/dp)

where x is distance from the fiber core, starting at x = 0 at the core-cladding interface, E0 is the

magnitude of the field at the interface, and dp is the penetration depth.

The penetration depth is given by

where is the wavelength of the light source, is the angle of incidence of the light at the core/cladding

interface, nco and ncl are the refractive indices (RI) of the core and cladding, respectively.

Fundamentals

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The cross-section profile of an optical fiber cut along the long axis. The thick arrow represents onemode of light entering in multimode with an angle of incidence . Even though most of the light is

propagated, there is a small portion known as the evanescent field that decays to 1/e of its value at the

core-cladding interface at a distance ofdp. Typical SM fibers that operate at 13101550 nm have a core

of 8m and an overall diameter of 125m.

Fundamentals

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(A) De-cladded optical fiber. (B) Tapered optical fiber. (C) U-shape probe. (D) Tapered tip.

Fundamentals

Evanescent field absorption.

When light is transmitted through a tapered fiber and the evanescent field interacts with the analytes in

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When light is transmitted through a tapered fiber and the evanescent field interacts with the analytes in

the tapered region, the transmission decreases if the analytes absorb in the wavelength used for

transmission. The magnitude of the transmission decreases and depends on the analytes concentration. In

order to use tapers for absorption measurements, the light source must be at a wavelength which is

absorbed by the sample analyte. In a uniform-core optical fiber stripped of cladding, absorbance is

governed by the LambertBeers Law:

A = L

where is the absorption coefficient, L is the length of interaction, and is the fraction of light in the

.

The absorption coefficient is given by: = C

where is the molar absorptivity, and C is the concentration of the species. Sensitivity is given by:

In a uniform-core optical fiber, is quite low. Considering a fiber with a 200m diameter, is in the

104 range. Geometric factors which influence the sensitivity of an absorption TFOBS include radius,taper ratio, length, and bending.

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LITERATURE

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1

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Two probes were prepared under identical conditions and were placed in for 3 h in whole cell protein

extracts from stimulated cells. The probes were then rinsed with PBS containing 0.05% Tween-20and placed for 1 h in a solution of Alexa 430 labelled antibodies, specific to phosphorylated STAT3

(anti-PY-STAT3).

1

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2

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2

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Week 5 (27/10/2010)

Electrochemical Sensors

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Potentiometric Biosensors

Amperometric Biosensors

Impedimetric Biosensors

Week 6 (03/11/2010)

Massed Based Biosensors

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Acoustic Biosensors

Cantilever based Biosensors

Week 7 (10/11/2010)

Paper Presentaions

Week 8 (24/11/2010)

Other Optical Sensors Absorption spectroscopy, Fluorescence spectroscopy,

Luminescence spectroscopy, Internal reflection spectroscopy, Light scattering

Week 9 (01/12/2010)

Protein and DNA Microarrays/Biochips

Week 10 (08/12/2010)

Seminar Week

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Week 11 (15/12/2010)

Nanotechnology in Biosensors

Nanowire Biosensors

Nanotube Biosensors

Nanoparticles

FET and CMOS devices

Week 12 (22/12/2010)

Biosensors Market and Future

Documents

BM 519 WEEK 4