bioanalysis in nanotechnology era - upb...helps make up tendons, ligaments, cartilage and other...

BIOANALYSIS INNANOTECHNOLOGY ERA

- TRENDS AND DEMANDS -

Prof.dr.eng. Gabriel-Lucian RADU

30, January 2013 Bucharest

Bioanalysis is a progressive domain for which the future holdsmany exciting opportunities to further improve sensitivity,specificity, accuracy, efficiency, assay throughput, data quality,data handling and processing, analysis cost and environmentalimpact. Standards set by regulatory bodies regarding methoddevelopment and validation increasingly define the boundariesbetween speed and quality.

Bioanalysis is now involved with the discovery, measurementand qualification of pharmacogenomic profiles and biomarkersand, subsequently, the development of diagnostic kits toindividualize patient characterization and treatment.

What is Bioanalysis?

Bioanalysis is a sub-discipline of analytical chemistry coveringthe qualitative and quantitative measurement of xenobiotics(drugs and their metabolites, and biological molecules inunnatural locations or concentrations) and biotics(macromolecules, proteins, DNA, large molecule drugs,metabolites etc.) in biological systems.

Nanoscale Measurements

TypicalTypical NanosizesNanosizes of Cellular Speciesof Cellular Species

BiologicalSpecies Example Typical Size

(nm)Typical

MolecularWeight

Smallassemblies

Ribosome 20 (sphere) 105 – 107

Nucleic acids tRNA 10 (rod) 104 – 105

Small proteins Chymotrypsin 4 (sphere) 104 – 105

Large proteins Aspartatetrans-carbamoylase

7 (sphere) 105 – 107

DNA

mRNA

Proteins

Central Dogma of Life

Protection of DNA

Amplification ofgenetic information

Efficient regulationof gene expression

20 amino acids30,000 – 40,000 genes>2,000,000 proteins

• A cell is an organization of millions of molecules

• Proper interrelationship between these molecules isessential to the normal functioning of the cell

• To understand interrelationship:*Determine the arrangement of atoms (molecules)*

• “Chemical” biology follows the continuum (cellular,molecular, etc.)

Organ Tissue Cell Molecule Atoms

Structural Biology

Genomics DNA (Gene)

FunctionalGenomics

Transcriptomics RNA

Proteomics PROTEIN

Metabolomics METABOLITE

Transcription

Translation

Enzymaticreaction

The “omics” nomenclature

GenTranscriptProteMetabol

~ome Sequence of acomplete set of

GenesTranscriptsProteinsMetabolites

=

GenProteMetabol

~omics = Analysis ofthe

GenomeProteomeMetabolome

A few definitions

BioanalysisAdvances in instrumentation allow for more rapid and precisecharacterization of complex biological systems. Quantitativeinformation is essential in the anaylsis of a (bio)chemical system.

ComputersProteomics

BioinformaticsGenomics

Results

Experiment

Protein QuantificationChemical Sensors

Compound Identification

Capillary ElectrophoresisMicroarrays

Chromatography

Mass Spectrometry

Instrument Design

Disease Markers

Composition => Structure => Function

Genome SequencingGenome Sequencing•• ShotgunShotgun•• DirectedDirected•• Venter / CollinsVenter / Collins

ProteomicsProteomics•• ““ClassicalClassical””•• NewNew

PostPost--TranslationalTranslationalModificationModification•• TopTop--DownDown•• BottomBottom--upup

RNARNA•• OligoOligo ArraysArrays•• Spotted ArraysSpotted Arrays

Measurement ScienceMeasurement Scienceat the Molecular Levelat the Molecular Level

BIOANALYSIS TODAY

The Human Genome Project

UnderstandingUnderstandinggene functiongene function

The function of aspecific gene canbe approachedfrom manyscientificperspectives witha variety of tools

Microarray scanners (RNA)Chromatography-Mass

Spectrometry(LC-ESI-MS)

Electrophoresis(DNA Sequencing)

What was measured?

From Chromosome to Sequence

Whole Genome Shotgun Sequencing

SequencingExperiments

SequenceAssembly

Genome with Unknown Sequence Reconstructed Genome Sequence

DNA Fragments

Sequence Assembly

INPUT

ACCGTGCGTGCTTACTACCGT

OUTPUT

TTAC----- -TACCGT-- --ACCGTG- ----CGTGC TTACCGTGC

Assembly

Sequence Variability

The Functional genomics era

HumanGenomeSequence

Tailored Drug&Diagnostics

Expression Variability

Phenotypicvariation

Polymorphism (SNPs)

MolecularPhenotype

Post-genomic era

Genome

Geneexpression

Proteins

Metabolism

Metabolomics

Proteomics

Transcriptomics

Genomics

Genotype

Environmentalstressors

PROTEOMICS

Proteins form the structural framework of tissues andother structures in the body. For example, collagenhelps make up tendons, ligaments, cartilage andother connective tissue.

Proteins carry substances throughout the body. Forinstance, hemoglobin is a carrier of oxygen andcarbon dioxide.

Proteins work as enzymes to facilitate chemicalreactions. There are over 1,000 known types ofenzymes in the human body.

The proteins myosin and actin, allow muscles tocontract.

Proteins regulate physiological processes and controlgrowth.

Proteins are an integral part of the immune system.Antibodies, for example, consist of a type of proteinused by the immune system.

Why Proteins?

Proteins

Hierarchy of structures

1° 2° 3° 4°

Sequence / AssemblyPackaging

The Basics – protein structureprimary structure – amino

acid sequence

DRLEFIVTALLKPW

N-terminus C-terminus

tertiary structure – proteinfolding

http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

quaternary structure –multimeric complexes

http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

secondary structure – localspatial arrangement

http://mcl1.ncifcrf.gov/integrase/asv_secstr.html

-sheet -helix

Proteins classes for bioanalysis

Membrane Soluble proteins Nuclear Chromosome-associated Phosphorylated Glycosylated Complexes

Proteomics

- Every gene product expressed (>1 copynumber) at a given time, in a given cellular state

What information about proteinsdo we want ? Identity Absolute abundance Relative abundance Post-translational state Biochemical activity (function) Non-covalent associations in vivo half-life Sub-cellular localization

An organism’s proteome: a catalog of all proteins expressed throughout life expressed under all conditions

The goals of proteomics: to catalog all proteins to understand their functions to understand how they interact with each

other

Analytical definition of proteomics:

Identity, quantity and function of all theproteins in a mixture

General flowfor proteomicsbioanalysis

SEPA

RA

TIO

NID

ENTI

FIC

ATIO

N

Molecular MS Applications: Proteomics

Proteome: The group of proteins related to a celltype (with a certain genome) under certain conditions(often forced on the cell)

Genome: The complete DNA sequence of a set ofchromosomes.

Proteomics: The analysis of native and post-translationally modified proteins to characterizecomplex biological systems. There are at least three“types” of proteomics: Profiling Proteomics: Identify the proteins in a

biological sample (or differences between proteins inmultiple samples)

Functional Proteomics: Determine protein functionsby finding specific functional groups or interactions

Structural Proteomics: Determine the tertiarystructure of proteins and their complexes.

Molecular MS Applications: Proteomics “Peptide mass mapping”:

used to ID proteins bycomparison to a database.

Accurate mass methods(single MS stage) areusually used, followingdigestion by an enzyme(e.g. trypsin) that “chewsup” the peptide intofragments.

The better the massaccuracy, the less chanceof isobaric (same mass)interferences.

Generic mass spectrometry (MS)-basedproteomics experiment

LC/MS Instrument Basics:Single MS- Primarily used as a detector

Ion Source Interface Mass Analyzers Detection

-ESI-APCI-APPI

-Orifice – Sk-Capillary

(Hot or cold)

1- Quadrupole2- Ion Trap (4 types)3- Time of Flight

CEMDiscrete dynodeCCDPhotomultiplier

LC

MS System – under vacuum

Protein identification by MS

Artificialspectra built

Artificiallytrypsinated

Database ofsequences

(i.e. SwissProt)

Spot removedfrom gel

Fragmentedusing trypsin

Spectrum offragmentsgenerated

MATCH

Libr

ary

Ionization techniques – Application range

Electrospray

APPI

MS Analyzer comparison- Mass AccuracyQuadrupoles

TOF AnalyzerLinear ModeReflectron Mode

Common Ionization Methods for TOF MSMALDIESI

Sample Application

‘Time-of-Flight’ Mass Spectrometry

Simplified Schematic- TOF-MS

The analyser, detector and ionisation source areunder high vacuum to allow unhindered movementof ions

Operation is under complete data system control

analyzer

Time-of-Flight (TOF)

drift tube

Sample Applications (TOF MS)

Biomarker Discovery

Determination of Sites of Glycosylation inGlycoproteins by QTOF MS

200 400 600 800 1000 1200 1400 1600 1800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800mass0

100

%

366.17

204.10

292.13

3151.41

2989.36

2988.521270.59657.30

547.22

1173.85731.38 1049.51

820.33968.60

2786.232096.961577.15

1496.80 1577.88 2079.991584.721759.86

2624.18

2098.08 2549.112787.33

3516.643152.56

3153.37

3515.433441.523354.58

3166.71

3517.54

3808.753518.78

3529.693645.67

3821.45

3822.85

[Peptide+HexNAc]+NeuAc

Hex

Hex

HexHexHex

HexNAc

HexNAcHexNAc

VENHTACHCSTCYYHK

Hyphenated Ion Methods

MALDI-ion mobility-orthogonal TOF MS (MALDI-IM-oTOF) Used to study biomolecular structure Detection limit approaches conventional MALDI-MS

MALDI-IM-oTOF experiment can simultaneouslygive mass spectra and molecular“conformation” (size and overall shape)information on desorbed ions.

Applications: mixture analysis, proteomics,analysis of complex tissues and micro-organisms.

The Mechanism of MALDI(Matrix-Assisted Laser

Desorption/Ionization):Ion Desorption The Formation of a ‘Solid Solution’ Matrix Excitation Analyte Ionization

Ionization Methods —MALDI

MALDI TOF Mass Spectrometry

Tandem MS of N-Glycans Derived fromRibonuclease B

9.0 271.6 534.2 796.8 1059.4 1322.0Mass (m/z)

0

10

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30

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OHO

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CCH3

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OH

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CCH3

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OH

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CH2OH

OH OH

OH

3,5X4’

0,40,4AA33

3,53,5AA33

3,5 A

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3,5A53,5X4”

3,5X3

1,51,5XX33

1,4A5

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1,5X3

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Na+

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MALDI-TOF Ex. Data

What can a QTOF do for you…?

Genome,Proteome,now what?

Metabolome!!!

Metabolites Intermediates and products of metabolism Examples include antibiotics, pigments, carbohydrates,

fatty acids and amino acids Primary and secondary metabolites

Metabolome Refers to the complete set of small-molecule

metabolites, is the metabolic complement (metabolitepool) of a cell, tissue or organism under a given set ofconditions

Metabolomics The study of the metabolome Newly emerging field of 'omics' research Comprehensive and simultaneous systematic

determination of metabolite levels in the metabolomeand their changes over time as a consequence of stimuli

From metabolites to metabolomics

Metabolite complement of a proteomeVariable

In different cell and tissue types in sameorganism

In different growth and developmentalstages of organism

Dynamic Depends on response of genome &

proteome to environmental factors Disease state Drug challenge Growth conditions Stress

The Metabolome

Factors influencing the metabolome

Tissue or biofluid sampleTissue or biofluid sample

Measure the metabolite profileMeasure the metabolite profile

Treat profile as‘fingerprint’ for

classification purposes

Treat profile as‘fingerprint’ for

classification purposes

Explore profile to gainmechanistic insight intothe biological response

Explore profile to gainmechanistic insight intothe biological response

Statisticalbioinformatic tools

Bioanalytical tools

(applied/clinical) (basic research)

1. Mass spectrometry2. 1H NMR spectroscopyEtc.

Bioanalysis

What can we do with metabolomics

Metabolomics enables: Qualitative and quantitative display of

metabolite concentration patterns Assessment of global changes of conditions Comparative analysis of (bio)samples Provides information from which

biological systems may be influenced

Drug assessmentClinical toxicologyNutrigenomicsFunctional genomics

Main ApplicationsMain Applications

ForensicsForensics•Medicine - drug discovery, drug design

EcologyEcology• Why can invasive species succeed in some environments & not in

others?Testing of GMO materialsTesting of GMO materials• How has transgenic manipulation changed food characters?

Pathway discoveryPathway discovery• pathway discovery• signal character of metabolites• the function is paralogous genes (enzymes)• what controls flux through a pathway?• exchange of metabolites in symbiosis• global change biology and metabolite change• YFM [your favorite mutant]• finding “new” enzymes – pathway engineering

““Systems BiologySystems Biology”” objectivesobjectives• Integrated knowledge of (plant) life on earth

Applications of metabolite analysis

Integration of metabolomics with other‘omics’ fields

Integrating genomics and metabolomics for engineering plant metabolicpathways (2008).

Proteomic and metabolomic analysis of cardioprotection: Interplaybetween protein kinase C epsilon and delta in regulating glucosemetabolism of murine heart.

Recent studies (2010) to integrate transcriptomics, proteomics andmetabolomics in an effort to enhance production efficiency understressful conditions of grapes.

Nutrigenomics is a generalised term which links genomics,transcriptomics, proteomics and metabolomics to human nutrition.

The name metabolomics was coined in the late 1990s (Systematicfunctional analysis of the yeast genome).Many of the bioanalytical methods used for metabolomics havebeen adapted (or in some cases simply adopted) from existingbiochemical techniques.Human Metabolome project – first draft of human metabolome in2007.

Nanotechnology Applicationsin Life Sciences

NanobiotechnologyCM Niemeyer, CA Mirkin

BionanotechnologyDS Goodsell

nanotechnology is used to createdevices to study biological systems .

e.g: new medical technologiesinvolving nanoparticles, deliverysystems or as sensors

study of how the goals ofnanotechnology can be guided bystudying how biological "machines"work and adapting these biologicalmotifs into improving existingnanotechnologies or creating new ones.e.g:Bionanotechnology

1.DNA Nanotechnology2.Cellular engineering

Anticipated MarketAn

ticip

ated

Fea

sibi

lity

Health and Life Sciences 2020

Water, Food, Bio-fuels 2020

Antic

ipat

ed F

easi

bilit

y

Anticipated Market

Trends in Bioanalysis

Control, improvements in living organismsBio-sensing at the micro and nano level, micro

and nano electromechanics Integration with wireless, RFID, photonics-

molecular level camerasTissue engineering, artificial organs, implants

and prothesesTargeted drug delivery and use of in vitro

capacitiesRapid scaleable bio-assays for molecule ID,

medical diagnosis and forensics Personalized medicine using large data sets of

patient information, disease statistics, genesequences and genotypes

Molecular recognition –targeted drug delivery toorgans, tumours - lab on chip

Converging Technologies by 2050?

1. "Clean Coal" technologies (science incubator)2. Bio-nano-health Monitors (application developer)3. Implantable Nanoarrays for Livestock (application

developer)4. "CO2 Sequestration" technologies (application developer)5. Environmental nanobiosensors (producer/application

developer)6. On-time Nano-vaccinology (technology developer)7. "Biomass Biofuels" technologies (application

developer)8. Medical "Tricorder" (producer)9. Smart Agri-bio Nanoencapsulation (tech. developer)10. Food-tracking Nanotags (science incubator)11. Directed Evolution Chips (technology developer)

Desirable applications of nanotechnology

“Smart” therapeutics Targetted molecular imaging agents

Nano-enabled consumer productsTissue engineeringBiological sensors/diagnostic tools

Trends: Biomolecular Medicine

Comprises several fields: Genomics, proteomics, computational protein folding,

molecular genetics, molecular immunology,computational protein folding, etc.

Short-term effects on EM probably moderate, comparedto disciplines like oncology, hematology andrheumatology

Long term effects are critical: Molecular adjuncts to resuscitation Molecular/genetic diagnostics Rapid protein repair Early prevention of cell-death triggers

Nanotechnology applied

Advantages of Nanoscale devices in Medicine• Devices smaller than 50 nm can easily enter most cells• Devices smaller than 20 nm can transit out of blood vessels• Devices are capable of holding thousands of small molecules

• Contrast Agents• Drugs

Major Areas of Development of Nanomedicine• Prevention and control

• Early detection

• Imaging diagnostics

• Multifunctional therapeutics

Trends: Drug Delivery

Organ/tissue-specific delivery systems Ability to deliver engineered or recombinant proteins to

tissues will be key Targeted tissue delivery will be a major step forward

innovations in interventional radiology Computational protein folding recombinant proteins, eg, active group + targeting

domain engineered virions nanotechnology engineered microorganisms antisense RNA technologies micelles, microspheres

Therapeutic delivery of drugs

Small scale “pills” can be taken up by cells Adding of antibodies can be used to target sick cells Administered through IV, the skin, inhaled, orally

Nano carrier for the delivery of proteins

Once injected in the body, the nanoparticles release the captured-drugs in acontrolled manner and over an extended period of time. Both processes(capture and release) are non-denaturing, which preserves structural integrity- and hence the biological activity - of the drug.

Biodelivery using protein-coated nanowires

•siliconcarbide

Nanopharmaceuticals

Liposomal formulations are the firstNanoPharmaceuticals introduced tomarket, formulation for doxorubicin isthe first product based on liposomes.Theses liposomes are called as“Stealth” liposomes with size<200nm which are long circulationwith hydrophilic (PEG)surface. These long circulatingliposomes found to target totumor tissue by a mechanism knownas enhanced permeation andretention (EPR). Hence liposomalformulation of doxorubicinconsiderably reduced the cardio-toxicity of drug. Many lipososomalproducts are under various phasesof clinical trials, here is the tableshowing some of product which arein market:

Liposome construction

Phospholipid Bilayers

Phospholipid

Variety of lipid structures

Non-viral gene therapy: Nanoparticles

Cell Membrane: 6-10 nm thick Micelle: Complexatin and condensation of oppositely-

charged polyelectrolytes. Can slip past cell membrane DNA-Chitosan Can be further functionalised for targetting specific cells

Trends: Artificial Organs

Biodegradable TissueScaffold

Inoculate withtissue-specific

cultured cells orstem cells

Artifical kidney

Continued progress with mechanical organs and tissues,especially pumps (hearts), tubes (vascular, ducts), joints, boneand muscle. New materials and processes

A new generation of artificial tissues and organs, representing aconfluence of several technologies

Bone cells grown on carbon nanotubes

Bone cells can grow and proliferate on a scaffold of carbon nanotubes. Scientistsfound that the nanotubes, 100,000 times finer than a human hair, are an excellentscaffold for bone cells to grow on.

Multifunctional magnetic nanoparticles

Combine various components

Multiple bio-applications

Bioseparation and cell biolabeling usingnanomagnets

5.34 nm

•Fe/Fe oxide nanocluster

Biomimetic Systems

Fabricating magnetically actuated nanorod arrays as biomimetic cilia

Nanotechnology includes Nanomaterials Any material that has nano-scale features are termed a nanomaterial

NanowiresNanowires

NanoparticlesNanoparticles

NanomembranesNanomembranes

NanoNano--othersothers

Are Nanomaterials New?

Milk (human milk and colostrum) casein micelles (50-300 nm) whey proteins (4-6 nm) lactose (0.5 nm) fat globules (300 nm)

Nanotechnology can be used for tissue engineering To grow a cell needs to adhere and spread Nanomaterials can navigate cell growth Cells can adhere to nanomaterials more strongly

Nanomaterials can change cell behaviorStem cells can be grown on nanomaterialsThe differentiation of the stem cell can be changed with nanomaterial

interactions

Bio-Nano Convergence

Silicon Nitride Nanopore

Silicon Nitride Nanopore for sequencing of DNA

Nano Opportunities:Targeted Heavy-Metal Binding

Utilization of a non-toxic polymer to bindheavy metals like arsenic in water or soil

Elastin Domain Metal Binding Domain

Fine tune affinitywith differentbinding sequence

Fine tune T by controlling amino acidsequence and no. of repeating unit (VPGXG)n

Clinical Proteomics: Goals

Develop new biomarkers for disease diagnosisand early detection

Identify new targets for drugs

Better evaluate the therapeutic effect ofpossible drugs

Clinical Proteomics

Find changes in: Cell or tissues Subcellular

structures Protein complexes Biological fluids

Analyze theproteome of

bothdiseased andhealthy cells

“Science is built up with facts, as a house is withstones. But a collection of facts is no more ascience than a heap of stones is a house.”

Jules Henri Poincaré

electroanalysis

research groups

biosensingclinical biochemistry

extraction&conditioning

Main research area: Analytical chemistry devoted toBiological Compounds, Bio-Products and Bio-ProcessesAssessment

National Institute of R & D for Biological Sciences

Centre of Bioanalysis

History

1991 Laboratory of Chemistry and Biochemistry -Institute of DevelopmentalBiology

1996 Department of Analytical Biochemistry – National Institute for BiologicalSciences

Department of Bioanalysis -National Institute for Biological Sciences

Centre of Bioanalysis –National Institute for Biological SciencesCentre of Excellence – BIOTECH National Program

20032002

2005

Centre of Bioanalysis - National Institute for Biological Sciencespresent

Main research directions,competencies

Development and validation of analytical methodsfor (bio) compounds and bioproducts assessment

High performance liquid chromatography (including 2D HPLC) –Diode Array-FL- Mass Spectrometry/MALDI based methods forassessment of

Blood samples (SAM/SAH; antibiotics; vitamins)

Algal cultures (carotenoids; xanthophylls)

Overlaid HPLC-DAD chromatograms forxanthophylls from 4 strainsChlamidomonas Reinhardtii

Overlaid HPLC-FLchromatograms for SAM&SAHstandard and blood sample


Bio-mimetic materials development,characterization and application

Low-density lipoprotein and Phosphatidyl-cholinebased biomimetic systems applied in Antioxidant capacity assessment of compounds with potentialantioxidative effects Assessment of ROS toxicity and mechanism type

Functionalized nanoparticle supportsas potential carriers for active substances

LDL MALDI ToFanalysis

98,498,698,8

9999,299,499,699,8100

0 20 40 60 80 100time, days

% r

elea

se00,20,40,60,811,21,41,6

% lo

ss w

eig

th

release profile

loss weigth

Serin-protease inhibitory releasefrom CHIT-magnetic NP carriers


Electrochemical methods environment: heavy metals, pesticides;

antibiotics; other EDC

Electron transfer processes assessment

LDL- modified ITO electrodes

SW voltamograms Cu(II); Cd (II)fortified water samples

a

b

FTIR based methods for nano-composite, modified surfacesand polymers/polymer supports etc. characterization


3525

.02

3385

.99

2882

.70

2874

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1659

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1553

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1409

.19

1385

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1066

.84

1065

.90

662.

8162

1.37

451.

96

500100015002000250030003500Wavenumber cm-1

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ittan

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]

C:\Documents and Settings\Mira\Si_chit.3-str.0 9/12/2009 4:26:46 PM

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FTIR spectra chitosan functionalized NP

Main research directions, competencies

Biosensing systems development for activeprinciples and contaminants determination

Enzymatic biosensors Environmental analysis (pollutants: PCBs; heavy metals; phenols) Food analysis :

nutraceuticals: polyphenols; contaminants: PAH, acrolein; toxin

Whole cell biosensors (PSII-NWFET sensors)

Research Infrastructure (1/3)

HPLC devices

2D-HPLC –UV-Vis-FL-Accuspot HPLC – DAD - MS


Spectrochemical devices

FTIR spectrometer

UV-Vis-NIR-FL spectrometer(optical fiber) MALDI-ToF

spectrometer

Electrochemical devices


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