Lecture:

Bioanalysis: Arbeitstechniken in der biochemischen Analytik

o. Univ. Prof. Thomas Schalkhammer   TUDELFT/ Vienna Biocenter/FH-Wiener Neustadt

‘hand out'

 

1. Electro-optical properties of molecules

 

1.1 Absorption of light

 

Basics: Optics

Spectroscopy of molecules

Bathochromiceffectofsolvents,

Conformationandpeptideabsorption,

Effectofsolventonabsorptionspectra,

Elektromagneticwaves,

Hypochromiceffects,

Hypochromy,

LambertBeerslaw,

Matrixeffects,

Spectroscopicpropertiesofchromophores

 

Basics: Polarisation of light

Linear and circular dichroism

LinearDichroism,

Opticalrotationdispersionandcirculardichroism

 

Basics: Molecule vibrations

Infra red spectroscopy

Infraredspectroscopy,

RulesofselectionforIRandRaman,

Spectroscopyinthenearinfrared

 

Basics:  Scattering

Raman spectroscopy

EnergyschemeforRamanspectroscopy,

Ramanandproteins,

Ramanandproteins,

Ramanspectroscopy,

Ramanspectroscopy2

 

1.2 Emission of light

 

Basics: Light emission

Fluorescence spectroscopy

Activatedfluorescencelabels,

AttenuatedTotalReflectionFluorescenceImmunoAssay,

Concentrationdependenceoffluorescence,

Correctionoffluorescencespectra,

Dependenceoffluorescencefromc,

Effectofsolventsonfluorescence,

Energytransferbetweenfluorophores,

Fluorescencequenching,

FluorescenceenergytransferinDNAanalysis,

Fluorescentprobes,

Howtomeasurediffusion,

Intracellularmarkersforions,

Localisationoffluorophores,

Measuringtechnology,

Setupofthecuvetteinafluorimeter,

Quantumyield

 

Chemiluminescence

 

1.3 Magnetic properties 

 

Basics: Magnetism

The nucleus: Basic NMR and 2D-NMR

Lamoreffect,

Magneticdipoltransfer,

NMRtechnology,

NMRexample,

NMRovomucoidefromturkey,

NMR2D,

NuclearOE,

SolventsuppressioninNMRspectroscopy

The electrons: Electron spin resonance (ESR)

ESRtechnology,

Electronspinresonance,

Hyperfinestructure

 

 

 

2. Single molecule techniques

 

2.1 Under ambient conditions

 

Basics: Atoms

STM-Surface tunneling microscope (for conducting surfaces)

ScanningTunnelingMicroscopy,

SetupofaSurfaceTunnelingMicroscope,

STMpicturesofatomsInSbandDNA,

Vibrationdampingoftunnelingmicroscopes

AFM-Atomic force microscope (for any rigid surface)

Intermolecularforces,

SetupofAtomicForceMicroscope,

Principleoftunnelingmicroscopy

 

2.2 Under UHV conditions

MS-spectroscopy (for small molecules to some macromolecules)

Massspectrometry

 

 

 

3. Peptides

 

Sequencing of proteins

Bromcyanbasedcleavageofproteins,

CleavageofNterminalCaps,

DABIAbasedlabelingmethod,

Edmancleavagetipsandtroubles,

Edmancleavage,

Enzymaticdigestion,

Gasphasesequencing,

InhibitionbySDS,

Manualsequencinginbatchsystem,

Sequencingofproteins,

Sequencingofproteins,

Carboxyterminal,

Cleavage,

Immobilization,

Yield,

SScounting,

AmineCounting

Exampleofpeptidesequ

 

Peptide synthesis

Peptidesynthesis,

PeptidesynthesisusingFmoc,

PeptidesynthesisMerrifield

 

 

4. Biological surfaces and binding

 

Basics: Adsorption

Assays and stains for DNA, RNA, proteins, glyko, activity, immuno, ink,

Opticalenzymaticassayforglucose,

OpticalenzymaticassayforascorbicacidwithMTT,

Signalamplificationbysubstratecycling,

BicinchoninicacidAssay,

Comparisonofproteinassays,

DNAAssay,

Mikrobiuretproteinassay,

Cellbasedassay,

 

Labeling of bio molecules via: enzymatic, optical, chemical, direct, indirect,

electrochemical, luminescent & chemiluminescent techniques

LabelingofDNA

Spectroscopy of small particles and colloids

ClusterResonance,

CLUSTER,

Mieresonanceofsmallmetalclusters,

Sodiumcluster,

Surfaceenhancedfluorescence

 

Basics: Surfaces & surface tension

Basis (Lipides, membranes,..)

PhasebehaviourofthelipidDPPC,

Structureoflipidmembranes,

 

LB-Technique

LBcurveforstearicacid

ChemicalBasisof LBTechnology,

DynamicBehaviouroflipid

 

Basics: Electronics and cells

Patch-Clamp-Technique

Analysisofionchannels

 

 

 

6. Antibody based techniques

 

Basics: Biorecognition

Immune precipitation, immune electrophoresis,latex agglutination assays

Basics: Immuno techniques and assays

Immuno techniques

Antibodies,

SandwichELISA,

Couplingofantibodiestoenzymestoformaconjugate,

Dependenceofimmunetiterfromimmunisationprocedure,

Immuneelectrophoresis,

Immuneprecipitation,

ImmunisationusingMAP,

Immunisationusingpeptides,

Immunisation,

Immunogicpeptides,

Productionofantibodiesviageneticmeans,

Purificationofantibodiesviaepitopeaffinitychromatograp,

Tcellepitopes

Immuno assays (ELISA with chromogenic, fluorescent and chemiluminescent substrates)

Biorecognition,

ELISA,

IndirectELISA,

Nonradioactivedet1,

Nonradioactivedet2

 

6. Electrophoresis

 

Basics: Electricity

Analytical electrophoresis

Advantagesofthinnergels,

Blotting,

Discelectrophoresis

Gradientgelelectrophoresis,

Immuneelpho,

PolymerizationofPolyacrylamide,

Thehorizontalsystem,

TheoryofSDSelectrophoresis

Enzymaticdigestioningel

 

 

 

 

 

7. Transduction of molecular events

 

Basics: Biochips and biosensors

Optical biosensors

Surface Plasmon Resonance

Electrochemical Biosensors

Fluorescence Biosensors

Membrane Biosensors

Piezo and SAW,..

BiosensorusingNADH,

Directredoxtransferenzymeelectrode,

Immunoreactor,

Nanogravimetry,

Principleofredoxmediationinelectrochemicalsensors,

Reversibleimmunoassaybiosensor,

SPRtechnique,

SPR,

EELISA,

SproteinDNAChip,

Polypyrrolsensor,

GODsensor,

 

 

 

  This database is free for education - commercial use prohibited.

  It adds basic information to the lecture but is by no means complete.