1. Detection systems part 2

2. 1Introduction 2Theoretical background Biochemistry/molecular biology 3Theoretical background computer science 4History of the field 5Splicing systems 6P systems 7Hairpins 8Detection techniques 9Micro technology introduction 10Microchips and fluidics 11Self assembly 12Regulatory networks 13Molecular motors 14DNA nanowires 15Protein computers 16DNA computing - summery 17Presentation of essay and discussion Course outline

3. Scale

4. SEM and TEM

5.  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

6. Transmission electron microscope

8.  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 Transmission electron microscope

11. TEM of a plant cell

12. TEM of outer shell of tumour spheroid

13.  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

17. Contrast Incident Electron Beam Contrast formation

18. Ribosome

19. Ribosome with SEM

20. SEM of tumour spheroid

21. Scanning electron microscope

22. Fly head

27. STM and AFM

28. from http://www.di.com/  Characteristics of common techniques for imaging and measuring surface morphology Scanning probe microscopes

29.  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

30. Type Properties used for scanning ResolutionUsed for STM Tunneling Current between sample and probe Vertical resolution < 1 Å *Lateral resolution ~ 10 Å => Conductors => Solids SP Surface profileVertical resolution ~ 10 Å *Lateral resolution ~ 1000 Å  Conductors, insulators, semiconductors => solids AFMForce 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 forceVertical 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 Scanning probe microscopes

31.  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 Scanning probe microscopes

32.  using a scanning tunneling microscope it is possible to image individual nickel atoms Scanning tunneling microscopes

33.  it is also possible to manipulate individual iron atoms on a copper surface Scanning tunneling microscopes

34. Iron on copperCarbon monoxide on platinum Scanning tunneling microscopes  it is also possible to have some fun

35. Xenon on nickel Scanning tunneling microscopes  it is also possible to have some fun

36.  With an atomic force microscope it is possible to image the carbon atoms of a carbon tube. Atomic force microscope

37.  Or manipulate carbon tubes. Atomic force microscope

38.  Or have some fun again. Atomic force microscope

39.  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 Scanning probe microscopes

40.  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 Scanning probe microscopes

41.  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 Scanning probe microscopes

42.  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 Scanning probe microscopes

43. G. Binnig, Ch. Gerber and C.F. Quate, Phys. Rev. Lett. 56, 930 (1986) First atomic force microscope

44. Atomic force microscope

45.  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 Atomic force microscope

46.  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 Atomic force microscope

50.  Micofabricated cantilever beams and probe tips for AFM. Atomic force microscope

52.  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

53. Atomic force microscope

54.  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 Atomic force microscope

55.  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

56.  nanopatterning of a growing number of molecular and biomolecular ‘inks’ on a variety of metal, semiconductor and insulator surfaces. NanoPen

57.  numerous applications are foreseen

58.  an AFM tip has been used to dissect a human chromosome to remove a specific gene NanoScalpel

59.  an AFM tip has been used to dissect a plant to remove a specific protein NanoScalpel

60. Experiment - AFM force spectroscopy Anselmetti, Smith et. al. Single Mol. 1 (2000) 1, 53-58 Nature - DNA replication, polymerization DNA unwinding

61. Surface Plasmon Resonance

62. angle Reflectivity Light (ω) 2D-detector array Surface plasmon wave (K sp ) Evanescent wave (K ev ) Surface plasmon resonance

63. Condition of Resonance = : Theory of surface plasmon resonance

64. Surface plasmon resonance

65. SPR angleReflective index Methanol63 o 1.329 Water66 o 1.34 Ethanol67 o 1.363 Hexane69 o 1.375 Surface plasmon resonance

66. (1) bare gold(2) immobilization (3) hybridization SPR on biochips (1) (2) (3) SPR Resonance Angle

67. Bryce P. Nelson, Anal. Chem. 2001, 73,1-7 Imaging SPR on biochips

68. http://www.gwcinstruments.com/ Imaging SPR on biochips

69. Robert M. Corn, Langmuir 2001, 17, 2502-2507 Imaging SPR on biochips

70. angle intensity SPR immuno sensor

71. (i) anti-progesterone (ii)anti-testosterone (iii)anti-mouse Fc SPR immuno sensor

72. Resonance Unit (RU): 1000 RU SPR angle: 0.1 degree Mass change : 1ng/mm 2 RI Change : 0.001 SPR binding kinetics: sensorgram

73. IBIS Technologies Commercial SPR systems

74. BIAcore SPR

76. Ellipsometry

77.  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

78.  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

79.  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

80.  Determine ρ = R p /R s (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

82.  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

83. Applications

84. Application

85.  Modified glass surface;  pattern biotin and avidin in perpendicular direction  use BSA to block the spaces avidin biotin Application

86. Gel electrophoresis

87. 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

88.  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

90.  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

91. For proteins or nucleic acid separation the gel itself is mainly composed of either agarose or polyacrylamid. Gels

92.  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

93. 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

94.  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

95.  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

96.  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

97.  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

98. Gel electrophoresis

100. PCR

101.  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)

102.  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 Polymerase chain reaction (PCR)