Detection systems part 2. 1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical background computer science 4History of the.

Detection systems

part 2

1 Introduction

2 Theoretical background Biochemistry/molecular biology

3 Theoretical background computer science

4 History of the field

5 Splicing systems

6 P systems

7 Hairpins

8 Detection techniques

9 Micro technology introduction

10 Microchips and fluidics

11 Self assembly

12 Regulatory networks

13 Molecular motors

14 DNA nanowires

15 Protein computers

16 DNA computing - summery

17 Presentation of essay and discussion

Course outline

Scale

SEM and TEM

electrons scatter when they pass

through thin sections of a specimen

transmitted electrons (those that do

not scatter) are used to produce

image

denser regions in specimen, scatter

more electrons and appear darker

Electron microscope

Transmission electron microscope


Provides a view of the internal structure of a

cell

Only very thin section of a specimen (about

100nm) can be studied

Magnification is 10000-100000X

Has a resolution 1000X better than light

microscope

Resolution is about 0.5 nm

transmitted electrons (those that do not

scatter) are used to produce image

denser regions in specimen, scatter more

electrons and appear darker




TEM of a plant cell

TEM of outer shell of tumour spheroid

No sectioning is required

Magnification is 100-10000X

Resolving power is about 20nm

produces a 3-dimensional image of

specimen’s surface features

Uses electrons as the source of

illumination, instead of light

Scanning electron microscope




Contrast

Incident Electron Beam

Contrast formation

Ribosome

Ribosome with SEM

http://cellbio.utmb.edu/cellbio/cit1.htm

SEM of tumour spheroid


Fly head

STM and AFM

OpticalMicroscope

SEM SPM

Sample operatingenvironment

AmbientLiquidvacuum

vacuum AmbientLiquidVacuum

Depth of field small large MediumDepth of focus medium small smallResolution: x,y 1 m 5 nm 0.1-3.0 nmResolution: z N/A N/A 0.01 nmMagnification range 1X -2 x 103X 10X - 106X 5 x 102 - 108XSample preparationrequired

little Freeze drying,coating

None

Characteristicsrequired of sample

Sample must not becompletelytransparent to lightwavelength used

Surface must notbuild up charge andsample must bevacuum compartible

Sample must notexcessive variationssurface height

from http://www.di.com/

Characteristics of common techniques for

imaging and measuring surface morphology

Scanning probe microscopes

Contact Mode AFM

TappingMode™ AFM

Non-contact Mode AFM

Force Modulation

Lateral Force Microscopy

(LFM)

Scanning Thermal

Microscopy

Magnetic Force

Microscopy (MFM)

LiftMode

Phase Imaging

Scanning Capacitance

Microscopy

Electric Force

Microscopy (EFM)

Nanoindenting/Scratching

(IMHO)

Scanning Tunneling

Microscopy (STM)

Lithography

Scanning techniques

Type

Properties used for scanning

Resolution Used for

STM

Tunneling Current between sample and probe

Vertical resolution < 1 Å*Lateral resolution ~ 10 Å

=> Conductors=> Solids

SP

Surface profile Vertical resolution ~ 10 Å*Lateral resolution ~ 1000 Å

Conductors, insulators, semiconductors=> solids

AFM Force between probe tip and sample surface (Interatomic or electromagnetic force)

Vertical resolution < 1 Å*Lateral resolution ~ 10 Å

=> Conductors, insulators, semiconductor => liquid layers, liquid crystals and solids surfaces

MFM

Magnetic force Vertical resolution ~ 1 Å*Lateral resolution ~ 10 Å

=> Magnetic materials

SCM

Capacitance developed in the presence of tip near sample surface

Vertical resolution ~ 2 Å*Lateral resolution ~ 5000 Å

=> Conductors=> Solids


using scanning probe microscopes it is

possible to image and manipulate matter on

the nanometer scale

under ideal conditions its is possible to

image and manipulate individuals atoms and

molecules

this offers the prospect of important new

insights in to the material world

this offers the prospect of important new

products and processes


using a scanning tunneling microscope it is

possible to image individual nickel atoms

Scanning tunneling microscopes

it is also possible to manipulate individual

iron atoms on a copper surface


http://www.almaden.ibm.com/vis/stm/images/stm16.jpg

Iron on copper Carbon monoxide on platinum


it is also possible to have some fun


Xenon on nickel


it is also possible to have some fun


With an atomic force microscope it is

possible to image the carbon atoms of a

carbon tube.

Atomic force microscope

Or manipulate carbon tubes.


Or have some fun again.


the scanning tunnelling microscope (STM) is

widely used to obtain atomically resolved images

of metal and other conducting surfaces

this is very useful for characterizing surface

roughness, observing surface defects, and

determining the size and conformation of

aggregates of atoms and molecules on a surface

increasingly STM is used to manipulate atoms and

molecules on a surface

Roher and Binnig won the Nobel Prize in 1986 for

their work in developing STM


a conducting tip is held close

to the surface

electrons tunnel between the

tip and the surface, producing

an electrical signal

the tip is extremely sharp,

being formed by one single atom

it slowly scans across the

surface at a distance of only

an atom's diameter


the tip is raised and lowered

in order to keep the signal

constant and maintain the

distance

this enables it to follow

even the smallest details of

the surface it is scanning

by recording the vertical

movement of the tip it is

possible to study the

structure of the surface atom

by atom


a profile of the surface is

created

from that a computer-

generated contour map of the

surface is produced

limited to use with

conducting substrates

this limitation was addressed

by atomic force microscopy

Logic gate


G. Binnig, Ch. Gerber and C.F. Quate, Phys. Rev. Lett. 56, 930 (1986)

First atomic force microscope


the atomic force microscope (AFM) is widely used

to obtain atomically resolved images of non-metal

and other non-conducting surfaces

this is very useful for characterizing chemical

and biological samples

increasingly AFM is used to manipulate

macromolecules and cells on a surface

Bennig, Quate and Geber are credited with

developing AFM and have received many major

awards


an AFM works by scanning a ceramic tip over a

surface

the tip is positioned at the end of a cantilever

arm shaped like a diving board

the tip is repelled by or attracted to the surface

and the cantilever arm deflected

the deflection is measured by a laser that

reflects at an oblique angle from the very end of

the cantilever





Micofabricated cantilever beams and probe

tips for AFM.



Contact mode imaging (left) is heavily influenced by frictional

and adhesive forces which can damage samples and distort image

data.

Non-contact imaging (center) generally provides low resolution

and can also be hampered by the contaminant layer which can

interfere with oscillation.

TappingMode imaging (right) eliminates frictional forces by

intermittently contacting the surface and oscillating with

sufficient amplitude to prevent the tip from being trapped by

adhesive meniscus forces from the contaminant layer.

Scanning modes in AFM


a plot of the laser

deflection versus tip

position on the sample

surface provides the

resolution of the hills and

valleys that constitute the

surface

the AFM can work with the tip

touching the sample (contact

mode), or the tip can tap

across the surface (tapping

mode) much like the cane of a

blind person.

proteins

bone cell


the NanoPen was developed by Chad Mirkin over

the past few years

a nanopatterning technique in which an AFM tip

is used to deliver molecules to a surface via a

solvent meniscus, which naturally forms in the

ambient atmosphere

NanoPen

nanopatterning of a growing number of molecular

and biomolecular ‘inks’ on a variety of metal,

semiconductor and insulator surfaces.

NanoPen

NanoPen

numerous applications are foreseen

an AFM tip has been used to dissect a human

chromosome to remove a specific gene

NanoScalpel

an AFM tip has been used to dissect a plant to

remove a specific protein

NanoScalpel

Experiment - AFM forcespectroscopy

Anselmetti, Smith et. al. Single Mol. 1 (2000) 1, 53-58

Nature - DNA replication,polymerization

DNA unwinding

Surface Plasmon Resonance

angle

Reflectiv

ity

Light (ω) 2D-detectorarray

p

nm800

x

Surface plasmon wave (Ksp)s

nmd 50 Evanescent wave (Kev)

z

x

mr

Surface plasmon resonance

*

*

sin

sin

2

2

pmr

pmr

s

ssn

smr

smrsp cK

*sin

pev cKCondition of

Resonance =:

Theory of surface plasmon resonance

50 55 60 65 70 750

20

40

60

80

100

120

140

160

Inte

nsi

ty o

f lig

ht [W

]

Angle [degrees]

Water Methanol Ethanol Hexane


SPR angle Reflective index

Methanol 63 o 1.329

Water 66 o 1.34

Ethanol 67 o 1.363

Hexane 69 o 1.375


(1) bare gold (2) immobilization (3) hybridization

SPR on biochips

(1) (2) (3)

SPR Resonance Angle

Bryce P. Nelson, Anal. Chem. 2001, 73,1-7

Imaging SPR on biochips

http://www.gwcinstruments.com/


Robert M. Corn, Langmuir 2001, 17, 2502-2507


angle

intensity

SPR immuno sensor

(i) anti-progesterone

(ii) anti-testosterone

(iii) anti-mouse Fc

SPR immuno sensor

Resonance Unit (RU): 1000 RU

SPR angle: 0.1 degree

Mass change : 1ng/mm2

RI Change : 0.001

SPR binding kinetics: sensorgram

IBIS Technologies

Commercial SPR systems

http://www.ti.com/

BIAcore SPR

BIAcore SPR

Ellipsometry

Allows us to probe the surface structure of

materials.

Makes use of Maxwell’s equations to

interpret data by Drude Approximation

Is often relatively insensitive to

calibration uncertainties.

Ellipsometry

Accuracies to the Angstrom

Can be used in-situ (as a film grows)

Typically used in thin film applications

Ellipsometry

html://www.phys.ksu.edu/~allbaugh/ellipsometry

Polarized light is reflected at an oblique

angle to a surface

The change to or from a generally elliptical

polarization is measured.

From these measurements, the complex index of

refraction and/or the thickness of the material

can be obtained.

Methodology

Determine ρ = Rp/Rs (complex)

Find ρ indirectly by measuring the shape of the

ellipse

Determine how e varies as a function of depth,

and thickness L of transition layer.

Theory

sincostantan ier

r i

s

p

Theory

Choose the polarizer orientation such that the relative

phase shift from Reflection is just cancelled by the phase

shift from the retarder.

We know that the relative phase shifts have cancelled if we

can null the signal with the analyzer

Null-ellipsometer

Applications

Application

Modified glass surface;

pattern biotin and avidin in perpendicular direction

use BSA to block the spacesavidin

biotin

Application

Gel electrophoresis

Electrophoresis is a technique used to

separate and sometimes purify macromolecules

that differ in charge, conformation or size.

Proteins and nucleic acids are mainly

concerned by that technique which is one of

the most used in molecular biology and

biochemistry (i.e. isozymes)

Electrophoresis

When charged molecules are placed in an

electric field, they migrate toward either the

positive (anode) or negative (cathode) pole

according to their charge.

Proteins can have either a net positive or net

negative charge (i.e. cathodic or anodic

peroxidases).

Nucleic acids have a constent negative charge

imparted by their phosphate.

Electrophoresis

Electrophoresis

Proteins and nucleic acids are electrophoresed

within a matrix or "gel". Commonly, the gel is

a thin slab, with wells for loading the sample.

Each extremity is in contact with an

electrophoresis buffer or the whole gel is

immersed within.

Ions present in the buffer carry the current

and maintain the pH at a relatively constant

value.

Electrophoresis

For proteins or nucleic acid separation the gel itself

is mainly composed of either agarose or polyacrylamid.

Gels

Agarose gels are extremely easy to prepare:

agarose powder is simply mix with buffer

solution, melted by heating, and poured.

Agarose is a polysaccharide extracted from

seaweed (non-toxic). The higher the agarose

concentration, the higher the resolution.

Low melting point agarose melts at about 65 C.

It is used to excised and purify fragments of

double-stranded DNA.

Agarose gels

Agarose gels have a large

range of separation but

relatively low resolving

power. By varying the

concentration of agarose

(from 4 to 0.5 %), fragments

of DNA, from 100 to 50,000

bp, can be separated using

standard techniques with a

resolution of a few bp.

Agarose gels

EtBr is a fluorescent dye that intercalates between

bases of nucleic acids and detection of DNA fragments

in gels.

It can be incorporated into agarose gels, or added to

DNA samples before loading to enable visualization of

the fragments within the gel, or present in a tank

were the gel is immersed after run and before

observation.

This last technique is the recommend because as might

be expected, binding of EtBr to DNA alters its mass

and rigidity, and therefore its mobility.

Ethidium bromide

Polyacrylamide is a cross-linked polymer of

acrylamide

The length of the polymer chains is dictated by the

concentration of acrylamid used, which is typically

between 3.5 and 20%.

Because oxygen inhibits the polymerization process,

they must be poured between glass plates (or

cylinders).

Polyacrylamide gels are significantly more annoying

to prepare than agarose gels.

Acrylamide gels

Acrylamide is a potent neurotoxin.

Disposable gloves when handling solutions

of acrylamide, and a mask when weighing

powder must be used.

Polyacrylamide is considered to be non-

toxic, but polyacrylamide gels should also

be handled with gloves due to the possible

presence of free acrylamide.

Acrylamide gels

Acrylamide gels have high resolutive power but

a relatively low range of separation.

Denaturing or not denaturing gel can be used.

First one are more resolutive, fragments of

DNA from 1 to a few hundred bp can be

separated with a resolution of 1bp. (Details

will be exposed during practical training)

Acrylamide gels

Gel electrophoresis

Gel electrophoresis

PCR

PCR is used to amplify (copy) specific DNA sequences in

a complex mixture when the ends of the sequence are

known

Source DNA is denatured into single strands

Two synthetic oligonucleotides complementary to the 3’

ends of the segment of interest are added in great

excess to the denatured DNA, then the temperature is

lowered

The genomic DNA remains denatured, because the

complementary strands are at too low a concentration to

encounter each other during the period of incubation,

but the specific oligonucleotides hybridize with their

complementary sequences in the genomic DNA

Polymerase chain reaction (PCR)

The hybridized oligos then serve as primers for DNA

synthesis, which begins upon addition of a supply of

nucleotides and a temperature resistant polymerase

such as Taq polymerase, from Thermus aquaticus (a

bacterium that lives in hot springs)

Taq polymerase extends the primers at temperatures

up to 72˚C

When synthesis is complete, the whole mixture is

heated further (to 95˚C) to melt the newly formed

duplexes

Repeated cycles (25—30) of synthesis (cooling) and

melting (heating) quickly provide many DNA copies



Detection systems part 2. 1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical background computer science 4History of the.

Documents

afm slide

sem slide

tem slide

ribosome slide

scale slide

plant cell slide

fly head slide

sem of tumour spheroid