DNA biochips & fluorescent bio-chip

1Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016

DNA biochips & fluorescent bio-chip

- Fluorescence, Uses in biology,

- Automatisation in biology, the example of "high throughput Screening"

- Principle of DNA chips / micro-arrays

-- Example : differential expression

-- Fluorescence reading apparatus

-- Fluorescence-based companies : domination of Affymetrix

-- Biotech example : hairpin probes

-- Innovation examples based on optics : Genewave, Genoptics










3

FROM GENE TO PROTEIN

Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016

Rib

osom

ePROTEIN

4

FROM GENE TO PROTEIN


Some jargon

SNP – Single Nucleotide Polymorphism

Denaturation : Separation of two DNA strands

PCR – Polymerase Chain Reaction

primers : amorces

5

Fluorescence in biology


- DNA (labelling does not impact function)

- proteins (much more delicate labelling, + use of GFP ,...)

- Labels : Dye type molecules (colorants) + quantum dots (CdS...)

The most sensitive biological detection (×100 vs. plasmons)

NB : SPR does not need labelling !










7

Fluorescence in biology


- DNA (labelling does not impact function)

- proteins (much more delicate labelling, + use of GFP ,...)

- Labels : Dye type molecules (colorants) + quantum dots (CdS...)

Biotechnology mastering the biochemical Lego … ensemble of exquisitely selective ligands … allowing , in fine, the obtainement of

... a signal~ No relationhsip with e.g. microelectronic technology

Will be illustrated later(hairpins)

strand to be detected(targed)

The most sensitive biological detection (×100 vs. plasmons)

8

Fluorescence in biology (techniques)


MULTIPLE TECHNIQUES ARE BASED ON FLUORESCENCE :

Titration : "end point measurement"

spatial tracking(resolution = /2nNA)

Fluorescence in situ hybridization (FISH)

Fluorescence resonant energy tansfert[donor acceptor] "FRET" echelle 1-10 nm

(De-)Polarisation of fluorescence(depends on the labelled molecule's ligand inertia, hence on hybridization)

Time-resolved (environment dependent decay, ~ µs typically)

●"How much ?" spectrofluorimeter, cuvette, precipitates, supernatant,...

●"Where ?" microscope (confocal), single molecule tracking, or more global

●"Near Whom ? " solutions containing "acceptors"( lower h) + microscope

●"With orientation memory ? " polarisation fluorescence analyser

●"At which pace ? " Time-resolved measurements ps ...ns.. µs (photophysics)

("real time" designates human time, 1s - 1 mn)


Biotechnology mastering the biochemical Lego … ensemble of exquisitely selective ligands … allowing , in fine, the obtainement of ... a signal

~ No relationhsip with e.g. microelectronic technology

...

~ ~ ~ for the time being,

Will you change your mind after you see microarrays, biochips, and "lab-on-a-chip" ?

"Techniques", "technologies", "biotechnologies"

semantic


DNA-biotin, S-HRP ,TSA (Cy3/Cy5)

B

Tyr Tyr

B

B

B

Tyr

B

TyrTyr

TyrTyr

Tyr

Tyr

TyrTyrTyr

AMDEX

B

TyrTyr

TyrTyr

Tyr

Tyr

TyrTyrTyr

AMDEX

BBB

B

B

BB

B

B

B

B

B

B

B


Signal amplification by biochemical means : example (tyramide)

Sensitivity without TSA : 0.5 fmolSensitivity with TSA : 0,01 fmol

DNA-biotin, S-HRP ,TSA (cy3/cy5)

B

Tyr Tyr

TSA Amplified Fluorescence Detection

Oligo-Biotinylated

Oligo-Cy3 Oligo-Cy5

Direct Fluorescent Detection

Oligo-Cy3 Oligo-Cy5

Oligo-Biotinylated

Green arrows: 10-7, 5x10-8, 10-8, 5x10-9 g/µl of oligo’s

Red arrows: 10-9, 5x10-10, 10-11, 5x10-12 g/µl of oligo’s


Cy3-Cy5 detection using 3Ddendrimer

3D 3D dendrimersdendrimers are are puchased from Genispherepuchased from Genispherebridging sequencebridging sequence

Labeled oligo (cy3 or cy5)

Bridging sequencing)










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Arrayed-sensor possible applications


HEALTHDiagnostic Pharmaco-genomicsSequencing?High throughput (drug] screening ("big pharma")

AGRO-ALIMENTARYtests are more and more mandatory

and numerous

ENVIRONMENTAir/water

DEFENSE (Bact., Chem., "biothreats")

(micro-array / microplate)


Parallelizing biology – season I – the microplate Ex : le criblage à haut debit(high-throughput screening)

96 wells

384 wells

… 1536 wells

8 carrouselseach 2 m high

These robots usuallywork 7/7, 24/24(Tecan, Cartesian,…)

chemical library200 000 to 1000 000 compounds

a few targets(proteinstightly related to pathologies)

At the heart of the robot

But still their [pixel size] >> 1 mm

8 x12=

A few thousand "hits"

A few "leads" : THE promising molecules


Parallelizing biology – season II – the microarray










PRINCIPLE of DNA CHIPS

Shrink unit size well below << 1 mmreduction of volumes and of reagents

particular ineterst of DNA : Measurementsmay take place ex situ on the dried material

PITCH ~ 200 to 400 µm,DIAM~ 100 to 200 µm

Support= microscope slide

25 75

1

And again more robots ...


A first robot prepares DNA sequences( on the spot or at a factory-lab)

glass slide

AC

GT

CG

CG

AC

GC

CG

CG

AC

GA

CG

CG

CG

A...

•• •• •• •• •• •• Oligo-nucleotides (< 100 bases) : tailored on demandcovalent binding to the slide most desired

•• •• •• •• •• •• •• •• •• •• •• •• Genomic DNA : obtained by biotechnologies (enzymes, ...) fromwhole cell DNA. This gives long fragments, of which selected parts are amplified (2^30 or more) by "PCR" (Polymerase chain reaction)(e.g. : fragment of yeast DNA (la levure)).Then, electrostatic binding may suffice ( Si-O-Si-alcane-H3N+) (ADN---)

•• Chemical treatment of the glass surface(control of charging state, of hydrophobicity, grating of functionalized groups

a process also know as "silanization")

•• "sticking" of DNA "spots" according to a deterministic schemeby robot [ or growth-on-the-spot by the Affymetrix method (see next)]


Some detail of surface chemistry

Standard glass substrate (25mm x 76 mm x 1 mm)

DNA

Electro-static coupling

NH3 NH3 NH3 NH3 NH3

+ + + + +

Amine coupling chemistry

(Case of long strands of genomic DNA)

(e.g. poly-lysine historically)


Surface chemistry and "silanization"

• epoxyde binding

• "silanisation" actually denotes the starting point (it often uses Si-NH2 chemistry):

DNA

CH

CH2

O

NH2 H-NH NH2

CH

CH2

NH2 NH NH2

OH

DNA Coupling- Attachement covalent

=> oligo DNA(25-100 -mer)

Si Si Si Si Si O

O O OO

HO

Hydrolysis+ condensation


Spotting : Robots, microplates, dedicated pins

Biorobotics BioGrid ArrayerContact Impression

Microplates 8x12 wells9 mm pitch

microarrays


Drop drying

Size of droplet : Compromise between surface tension and surface energyHydrophobic surface « high » droplet (small diam) ; hydrophilic surface « flat » droplet (large diam)


Combinatorial exercise

Pitch= 9 mm(96 wells)or half-pitch (384 wells)

.. and M plates

... for L slides

Let there be A pins (pin's pitch equals the pitch of microplate's well

with a 400 µm pitch

printing head with Pins

Microplate

Micro-array


Hybridization chamber

Liquid contains targetsintroduced on slide having probes at the surfaceControl of temperatureAgitation by syringes (through vials) or by other clever tricks

T control

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Then hybridization to analyte and reading (1/4)


glass slide

Fluorescent Labelling

"Analyte" DNA from RNA ex-pressed by stressed cells

N differentsequences

ROBOT + AstucesN' different sequences

Pre-hybridization rinsing


glass slide


Hybridization(mn … hoursliquid phase)




In a simple hybridization chamber(at 30-70°C to accelerate...)


glass slide


Hybridization(mn … hoursliquid phase)



Post-hybridizationrinsing (remove excess and

"nonspecific" adsorption)



glass slide


reading


glass slide

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Agitation / Beating Diffusion limiting steps


ArrayBoosterTM: Speed and Control for Micro ArraysIncubation chamber with integrated agitation based on 2D nanopumps

Surface Acoustic Waves are modes of elastic energy that can propagate at the surface of a solid body. Being the nanometer size analogon of earthquakes, they became industry over the last decade or so. SAW are usually used for radio frequency communication, acting as RF filter elements. They are easily excited on piezoelectric solids. Such materials deform if an electric field is applied to them. Rapid changes of such electric fields generated with an appropriate transducer are efficiently converted into a 'nanoquake on a chip'.

Both internal streaming as well as 'remotely controlled' actuation of the whole fluid volume can be achieved, depending on the power and wavelength of the SAW.

This way, smallest amounts of liquid can be actuated along fluidic tracks, different liquids can be combined, or efficiently mixed using the same technology


Don't tell ...

glass slide

The surface densityof DNA molecules grafted before hybridization… is random, sometimes vastly.

The DNA fraction resistant to rinsing is somewhere between 10% and 100 % (depending on stringency)

By and large, nothing is really constant ...BUT comparisons WITHIN A GIVEN SLIDE are (rather) conclusive.

(Inconsistencies have plagued the first biomedical findings and somewhat discredited the method, in the early 2000).


(Palaiseau/Evry)









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Fluorescent biochips : differential expression


Next : Optical signal & fluorescence

Fluorescent markers

Extraction of RNA/ reverse DNA transcription

Safe cells Stressed Cells

BIochips(selective hybridization selective)

Two different fluorophores , e.g. Green and Red

Measurement of the RED / GREEN ratio

fluorescent reading


Main fluorophores used for DNA chips

For DNA Biochips, two fluorophores are widely preferred

Cyanine 3 (Cy3)Exc. 532 nm, Fluo ~590 nm

Cyanine 5 (Cy5)Exc. 633 nm, Fluo ~670 nm

Why ? interest of large lambdas (less glass autofluorescence)but limited by increased sensitivity to photodegradation

Cy5 is already 10 times more sensitive than Cy3 to bleaching


Cy3: ~550 nmCy5: ~650 nm

DIFFERENTIAL EXPRESSION ASSAY (summary)

Yeast cells

extracted RNAmicroplates

microarray= biochip= DNA chip

PROBES(sondes)

TARGETS(cibles)


References

• Le livre "Microarrays" edited by M. Schena et al.

Various conference proceedings(formerly Chips-to-Hits, but has evolved)

a couple of tutorials on the web

http://www.bio.davidson.edu/courses/genomics/chip/chip.html











Confocality + fluorescence + PM

glass fluoresces : (little by unit volume)(× a lot of volume)

the "standard" fluorescence reading recipe


fluorescence spectra : absorption/emission

Alexa Fluor 514

400 500 600

emiexc

(nm)

Excitation Rejection !!!Rejection ratio > 106 to warrant the capability to see weak fluorescence sources


Rejection of fluorescence excitation

Filtres (here Chroma Inc.)

10-1

10-2

10-3

10-4

10-5

100

10-6

10-7

filtre interferentiel filtre complet

emission filter

excitation filter

500 600 700

600 700

emi.exc.

532 nm

Cy3

overall rejection ~108 à 109

But at the expense of a rather severe loss of fluorescence photons

large part of spectrum sacrificed by filter

because of small stokes shift+ high rejection required

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Quantum Dots : Monodisperse

Nanocrystalline Particles

(2-10 nm) made from

Semiconducting Materials


Improvement of fluorescent probes . . .

http://www.qdots.com/


Reading apparatus : biochip scanners

Present cost : about 20 to 50 K €...levelling but still going down

- Pseudo confocal microscopy(limiting the background glass fluorescencefrom below !)- mechanical X,Y scan of the slidemanty variantsThe slide mooooves along X alone, Mirror scanning of beams along Y axis"Flying objective" equiiping the far end of a periscope rotating around its optical axis- sequential scans of Cy3 et Cy5 (commutation of dichroic filters) (to be checked?)- two lasers- one or two PMT, whose voltage is selected (sensitivity vs. noise) according to the needs- (10 à 20 pixel)2 per spot, hence typically, 1 pixel is 10 µm or less- Collection lens aperture : NA~0.4 à 0.7 rather high apparently (get signal) but not too high (depth of field, limited flatness of slides !)


Commercial microarray scanners


CCD commercial scanners

Impossible d’afficher l’image.

uses CCD uses CCD


Sensitivity ("LoD")

Present detection threshold : about 100 molecules fluo/pixel

Important challenge !!!- Cost of reagents and need of large

samples

- for DNA : limiting the PCR of analyte (may distort)

- for expression – RNA (no "Copy-Paste"):Reducing biopsy's volumes, from about 1 cm3 nowadays, to only few mm3 (mouth epithelium on a swab)

•• Deposited drop ("spot")ca. (100 µm)3=10-6 cm3

•• [Cy]min ~ 4.6 10-11 mol/l~ few 1010 Cy /cm3

•• Hence there are 104 Cy /spotspread on 10x10 = 100 pixels

100 µm


Sensitivity (suite)

- Limitation of scan time to 300 s => a few tens of µs/point "intrinsic" pb of the sequential approach

(more physical approach : information retrieval is limited by the readout laser photon flux, however !) - Not exactly confocal lensing

(compatibility fast scan/large imperfect slides)

- Low extraction : Elementary calculation : from inside glass to NA=0.5 (au tableau)

- losses of optics and dichroic filters (as discussed)

- PMT limited QE at large wavelengths

- Convenient but "frail" Molecules (high yield and lack of chemical reactivity are not so compatible)

Why this limit ? ?? Isn't it commonly said that confocal detection allows down to single molecule detection ?? !!

Each molecule emits a large number of photons (106) under excitation before dying, doesn't it ?


An idea of the amount of data generated

Digital picture from your favorite compact : de 6 à 10 Mpix monochromes actually, CCD size is about 1/3" (=8.5 mm) diagonal to 1/2" (12.5 mm)

High end CC for 6x6 cameras (20 000 €) (?) : CCD size then reaches 36 x 43 mm (even larger than the former standatd > 24X36),

hence size ~<30 Mpix

Slide 24 x 72 mm scanned at a 10 µm pitch ~ 20 Mpix on 16 bits (dynamic range)=> CCD imaging is delicate without compromises

Anyway, for the biologists, there is an image treatment programwhich quantifies the data (segmenting to get spot contours, integrating in spots, etc.)After this step, biologists exchange files that "only " contain 10 K to 100K useful data (typicall two dozen data per spot, to check that they qualify for some quality tests)

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Photodetection : case of the PMT


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Photodetection : histogram


NyquistNoise +Poisson+Non ideality….

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Quantification of spots


Not a trivial issue

Background around spots : when does it become "the good reference" ? Its noise ? depends on (Cy3/Cy5) fluorescence channel ?

Spot : its limits ? how critical ? What should be removed?dusts , « hots spots » ?

If a spot is not homegneous enough, how valid is it ? What are the quality criteria ?


• Placement de la grille• Détourage des spots

(segmentation)• Quantification• Contrôle qualité

Analyse d’image

Surface = signal

Surface =bruit de fond local

F

B, B

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Quantification des spots


Problème delicat . . .

-Background autour des lames : Est-ce le bon ? Son bruit ? Suivant couleur ?

-Spot homogènes ? ronds ? poussière du fond ? - cf histo à forte tension du PMT : bruit (sur)-poissonien

=> apparence de « points chauds » qu’il ne faut pas tronquer

# occurences

# counts/pixel"bckgd" "signal"

1 decade

few decades

ideal

real, when good !

? via histogramme ? (histogramme "bimodal" ?)


- Fluorescence, Utilisation en biologie,

- Automatisation en biologie, l'exemple du "high throughput Screening"

- Principe des puces ADN

-- Exemple d'utilisation : l'expression differentielle

-- Appareillage de lecture de la fluo

-- Fluorescence & high-density : domination d'Affymetrix

-- Exemple de biotech : hairpins

-- Exemple d'innovation basees sur l'optique : Genewave

(Palaiseau/Evry)

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Affymetrix


Holds key patents until 2011on spotted chips, most notably on "high-density" chips, arbitrarily defined as more than (> 800(?) /cm2)

Format particulier

1 single fluorophore

1,28 cm2

inlet for liquid

16000 spots/cm2

Cost : ca . 3000 €/lame in 2002

CNRS was obliged in 2002 to subsidizefrench bio labs because the productwas so unique ...

200 000 chips sold in 2000

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Affymetrix


A A A A AA A A A A

A A A A AA A A A A

A A A A AA A A A A

A A A A A

A

AA

A

AA

A

Lithography technology( 1991) exploits parallelismSay A=0, C=1, all the 16 A/C variants of four basis are obtained in 4 steps only :1° 0101 0101 0101 01012° 0011 0011 0011 00113° 0000 1111 0000 11114° 0000 0000 1111 1111

Spot size ~decreased 25 µm x 25 µm to 5x5 !

"Genomic" chipsextracts of DNA of the whole genomeof e.g. Yeast(Saccharomyces Cerevisiae)

or a model plant, etc.Arabidopsis thaliana

Diverse offer

Chimie de photoactivation de l'attachement

masked parts

Protection for the next cycle

In situ Synthesis of 4N sequences in 4N steps

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Future : LAB-ON-A-CHIP


Silicon Lab-On-Chip for Integrated DNA Amplification and Detection

NanoChip® Electronic Microarray

The 12-centimeter diameter Gyrolab CD may look like a small disk. But it's a great lab.

Labo sur puce (evolves/Deries from the concept of µTAS=micro-TotalAnalysis System...)


Helicos : not all biochips are regular arraysComing soon, Human genome sequencingat less than 10 k$ ?

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Aequorea victoriaLa meduse... Verte

grâce à laquelle on a decouvert la"Green Fluorescent Protein"

un des segments de marche les plus prometteurs


FP INSERM et CNRS microscopie confocale

G.TRUGNAN INSERM U 538

St. Antoine, ParisS. BROWN CNRS

Gif-sur-Yvette

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GFP : une proteine fluorescente par transfert d'energie


Emission de la famille des GFPet derivees

HcRedEYFPEGFPECFPEBFP DsRed

Longueur d’onde (nm)

0

20

60

80

40

100

400 450 500 550 600 700650


R0 = distance qui produit un transfert de 50%

(2 à 6 nm)

r = distance reelle

FRET Fluorescence Resonance Energy Transfer

E= efficacite du transfert


Genewave


• Lame de verre :La fluorescence s’échappe vers le substrat ou n’est pas capturée par l’optique de collecte.

La plupart de la lumière est perdue

Collecte peu efficace Collecte facile et efficace

verre

air

verre

air

• AmpliSlide™: La fluorescence est exaltée et redirigéevers l’optique de collecte.

AmpliSlideDiagramme d’émission


Exemple de resultat de renforcement sur un test grandeur nature

(laboratoires de Gif s Yvette)


excitation

fluorescenceimagée

biopuce

optique de collection

capteur

80% pertes

Imageur grand champNA ~ 0.1 – 0.3

filtre

Microscopes confocaux à balayageNA ~ 0.4 – 0.7

excitation

fluorescenceimagée

biopuce


pinholePMT

80% pertes

séparatricefiltre

translation XY

Imagerie


excitation

fluorescenceimagée

biopuce


capteur

80% pertes

excitation

capteur

filtre

Imagerie conventionnelle,en champ lointain

NA < 0.3

Imagerie contactOptique ultra-simplifiée

Système miniature et robusteNA ~ 1.5

pixels

filtre

Optique de contactPrincipe


ApparatusResults

Software

Genewave Real-time tool HYBLIVE


Imagerie conventionnelle,en champ lointain

Imagerie contactOptique ultra-simplifiée

Système miniature et robuste

Comparatif


H7

H9N9MA

MB

H9

H2

H3

N1

H5

IVB

N2

H5N1

H5N2

H7N1

H9N2

H1

H3N2

H2N3H1N1

Fluorescence hybridization image showing typical patterns for Influenza A, H1N1 viral subtype.

Matrix of detected influenza strainsSource: CIRAD, Montpellier, France

Rapid, automated, multiplex typing of Influenza


Puces à fluorescence/ Fluorescent biochips : conclusion

- Rôle destiné à durer car le marquage fluorescent est un "système"

on "tagge" tout y compris les proteines (mais ...) . Cela sert dans beaucoup d'echafaudage de la biologie.

Long lasting role, fluorescence is “a system”, allowing to tag everything incl. proteins (but…). Used in

many“scaffolds” of biology

- Plusieurs goulot d'etranglement sont de nature optique et non biologique

: collection de la lumière, detection intelligente, traitement d'imageMany bottlenecks are of optical rather than biological nature : light collection, clever detection, image processing

- Appareillage automatise poussé dans le domaine des microplaques ;

- Appareillages miniature en cours (lab-on-a chip, labos sur puce)

A lot of highly automatized apparatus for microplates (96 wells), so much more coming for more miniature biochips

- Défi : la sensibilite ultime (~ 1 molecule ou 1 pathogène ou …)

Pas de limite physique profonde dans des temps de mesure raisonnables (~1s ou moins)

Challenge : the single molecule of single pathogen detection, no deep physical limits within few sec. meas. time

- Monde de grands industriels + start'up + grande importance de la Pte.Intellectuelle.

World of big industries + startups + high importance of intellectual property (IP)


Surface plasmon resonance (SPR) bioensors


Surface plasmon resonance (SPR) bioensors (2)


Role of the gold layer


DNA biochips & fluorescent bio-chip

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