Post on 11-Mar-2023
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
2Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Some jargon
SNP – Single Nucleotide Polymorphism
Denaturation : Separation of two DNA strands
PCR – Polymerase Chain Reaction
primers : amorces
5
Fluorescence in biology
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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 !
6Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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
7
Fluorescence in biology
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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)
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)
9Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
10Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
11Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
12Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Cy3-Cy5 detection using 3Ddendrimer
3D 3D dendrimersdendrimers are are puchased from Genispherepuchased from Genispherebridging sequencebridging sequence
Labeled oligo (cy3 or cy5)
Bridging sequencing)
13Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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
14
Arrayed-sensor possible applications
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)
15Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
16Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Parallelizing biology – season II – the microarray
- 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
17Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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 ...
18Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)]
19Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)
20Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
21Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Spotting : Robots, microplates, dedicated pins
Biorobotics BioGrid ArrayerContact Impression
Microplates 8x12 wells9 mm pitch
microarrays
22Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Drop drying
Size of droplet : Compromise between surface tension and surface energyHydrophobic surface « high » droplet (small diam) ; hydrophilic surface « flat » droplet (large diam)
23Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
24Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
25
Then hybridization to analyte and reading (1/4)
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
glass slide
Fluorescent Labelling
"Analyte" DNA from RNA ex-pressed by stressed cells
N differentsequences
ROBOT + AstucesN' different sequences
Pre-hybridization rinsing
26Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
glass slide
Fluorescent Labelling
Hybridization(mn … hoursliquid phase)
"Analyte" DNA from RNA ex-pressed by stressed cells
N differentsequences
Then hybridization to analyte and reading (2/4)
In a simple hybridization chamber(at 30-70°C to accelerate...)
27Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
glass slide
Fluorescent Labelling
Hybridization(mn … hoursliquid phase)
"Analyte" DNA from RNA ex-pressed by stressed cells
N differentsequences
Post-hybridizationrinsing (remove excess and
"nonspecific" adsorption)
Fluorescent Labelling
Then hybridization to analyte and reading (3/4)
glass slide
28Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
reading
Then hybridization to analyte and reading (4/4)
glass slide
29
Agitation / Beating Diffusion limiting steps
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
30Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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).
31Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
(Palaiseau/Evry)
- 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
32
Fluorescent biochips : differential expression
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
33Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
34Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Cy3: ~550 nmCy5: ~650 nm
DIFFERENTIAL EXPRESSION ASSAY (summary)
Yeast cells
extracted RNAmicroplates
microarray= biochip= DNA chip
PROBES(sondes)
TARGETS(cibles)
35Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
36Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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
37Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Confocality + fluorescence + PM
glass fluoresces : (little by unit volume)(× a lot of volume)
the "standard" fluorescence reading recipe
38Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
39Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
40
Quantum Dots : Monodisperse
Nanocrystalline Particles
(2-10 nm) made from
Semiconducting Materials
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Improvement of fluorescent probes . . .
http://www.qdots.com/
41Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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 !)
42Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Commercial microarray scanners
43Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
CCD commercial scanners
Impossible d’afficher l’image.
uses CCD uses CCD
44Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
45Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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 ?
46Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)
47
Photodetection : case of the PMT
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
48
Photodetection : histogram
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
NyquistNoise +Poisson+Non ideality….
49
Quantification of spots
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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 ?
50Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
• Placement de la grille• Détourage des spots
(segmentation)• Quantification• Contrôle qualité
Analyse d’image
Surface = signal
Surface =bruit de fond local
F
B, B
51
Quantification des spots
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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" ?)
52Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
- 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)
53
Affymetrix
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
54
Affymetrix
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
55
Future : LAB-ON-A-CHIP
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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...)
56Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Helicos : not all biochips are regular arraysComing soon, Human genome sequencingat less than 10 k$ ?
57
Aequorea victoriaLa meduse... Verte
grâce à laquelle on a decouvert la"Green Fluorescent Protein"
un des segments de marche les plus prometteurs
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
FP INSERM et CNRS microscopie confocale
G.TRUGNAN INSERM U 538
St. Antoine, ParisS. BROWN CNRS
Gif-sur-Yvette
58
GFP : une proteine fluorescente par transfert d'energie
Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
59Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
R0 = distance qui produit un transfert de 50%
(2 à 6 nm)
r = distance reelle
FRET Fluorescence Resonance Energy Transfer
E= efficacite du transfert
60Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Genewave
61Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
• 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
62Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Exemple de resultat de renforcement sur un test grandeur nature
(laboratoires de Gif s Yvette)
63Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
optique de collection
pinholePMT
80% pertes
séparatricefiltre
translation XY
Imagerie
64Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
excitation
fluorescenceimagée
biopuce
optique de collection
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
65Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
ApparatusResults
Software
Genewave Real-time tool HYBLIVE
66Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Imagerie conventionnelle,en champ lointain
Imagerie contactOptique ultra-simplifiée
Système miniature et robuste
Comparatif
67Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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
68Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
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)
69Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Surface plasmon resonance (SPR) bioensors
70Course Biophot. ESO3 : bio-chips, fluorescence, [SPR] , H. Benisty 1 février 2016
Surface plasmon resonance (SPR) bioensors (2)