Transcript Detection Methods

Screening Automation
Technologies
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Automatized HTS
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Need for automatization of HTS
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Advantages and problems
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Necessary equipment for automatization
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Detection methods in automatization
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Robotics
Need for Automatization
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Constant increase in library size
Need for Automatization
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24 targets/laboratorium in 2005
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G-protein koblede receptorer, kinaser, ionkanaler
Screening 792.000 samples against each target
Screening of 19 million samples/year/laboratorium (2005)
Increase of 20 % per year
50.000 samples/day (2005)
1 sample  1 datapoint  anourmous amount of data!
HTS  Ultra HTS: 10.000  > 100.000 samples/day
Important for Automatization
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Companies concentrate on
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Improvement of quality of data
Improvement of sucessrate (The magic triangle)
New technologies to achieve HT -> Ultra HTS
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New screening methods
Simply methods
Technologies for new classes of targets
The magic triangle of HTS
The optimization process for successful HTS (‘magic triangle of HTS’).The figure shows the key
success factors for modern lead discovery via HTS, namely time, costs, and quality. Optimal
lead discovery by HTS finds the right balance between these different elements.
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Target Classes (based on 2009)
Most of today’s leads finding efforts are focused on the left panel with the established drug classes . Future
drug discovery efforts might focus more on the novel target classes summarized in the right panel,
assuming that the given problems of proper readout technologies and suitable chemical space (‘drugability’)
can be overcome for these target classes.
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Advantages of automation
Faster
 Can screen larger libraries
 Fewer people involved (-> cheaper)
 Reproducibility of results (no human factor!!!)
 Improvement of safety
 Screening in optimal environment
 Miniaturization: less reagent and target, fewer plates
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The trend toward miniaturization in HTS
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Development of HTS
The figure illustrates evolution of throughput, costs, and resources (FTEs) invested in HTS
projects at Novartis from 1998 to 2007.
Entities are defined as single compounds per well; FTEs are defined as full-time employees
working on these HTS projects.
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Equipement for automation
Microtiterplates
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Standardization  Automation
Automation in HTS  more samples/plate
Number of samples
Working volume (µL)
96
100 – 300
384
5 – 100
1536
1 – 10
3456
0,2 – 3
Equipement for automation
Liquide handling tools
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Pin tools -> contact dispention
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V dependent on surface area of tips
Disadvantage: Variation in V,
contact
Advantage: simple, cheap, narrow
samples, small V
Equipement for automation
Liquide handling tools
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Air and positive displacement
(pipettes) -> non contact
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Small airspace required
Diadvantage: more expensive, larger V
Advantage: precise, broad working area
Equipement for automation
Liquide handling tools
Other non-contact dispensers:
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Peristaltic dispenser
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Capillary dispenser
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Piezoelectric dispenser
Equipement for automation
Liquide handling tools
Handling tools
Pin tools
Pipettes
Peristaltic dispenser
Capillary dispenser
Piezoelectric dispenser
Volume
5 – 500 nL
250 nL – 1 mL
> 1 µL
20 nL – 1 µL
0,5 nL – 10 µL
• Assay composition -> 2-5 components
Samples
Added Volume (µL)
96
5 – 100
384
5 – 50
1536
0,5 – 3
• Compounds: From stock solution in DMSO, <50 nL
Equipement for automation
Liquide handling tools
Problems with transfer of small volumes (nL):
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Surface tension, diffusion, evaporation
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Variations of the openings of the tips
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Viscosity (temperatur dependent)
Detection Methods
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Primary detection methods
 Fluorescens
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FLIPR (plate reader)
TR-FRET
FP (polarization spectr.)
FCS (correlation spectr.)
Luminescens
• Glow
• Flash
• Electrochemiluminescens
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Radiometric
• Filterbinding
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Absorbance
Detection Methods
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Plate readers
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Single point detection
using photomultiplier tubes
(PMT)
• high sensitivity
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Plates move under detector
Light source
• Tungsten
• Lasers
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• LED
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better sensitivity
cheaper
Specific wavelength
Wavelength
• Fixed
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Filtre used for specific
samples
• Variabel
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Multimode
• can read many detection
methods
• Advantage:
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Just one instrument ->
cheaper, less space
• Disadvantage:
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Lower sensitivity
Detection Methods
Detection Methods
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Plate Imagers
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Makro
• cooled (> - 100 grader)
CCD -> less background
• 2D picture
amplifier/photon counter
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both read out all samples
or at least part of the
plate simultaniously.
• Frame rate
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cooled CCD
• Requires longer
exposier/slower
”frame-rate”
photon counter
• fast ”frame-rate”,
but requires time
for processing
Detection Methods
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Plate Imagers
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Mikro
• High Content screening (HCS)
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Detailed information on molecular level
more images/sample
Advantages
• allows automatization of targets which were
difficult to screen
• Reduce costs for celle based assays
• Measure more things simultaniously (effectivity,
toxicity, specificity)
• optimization bedre understanding of ”false
positives”
Disadvantages
• Requires storage room and good software
Detection Methods
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Plate Imagers
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Mikro
• HC detection
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Laser scanners
• Original:
fluorescens cell
sorting
• Reconstitute
pictures (pseudo)
• God for whole cell
and bead analysis
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CCD imagers
• Original:
fluorescens
mikroskop
• can zoom in -> with
high resolution
Detection Methods
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Others
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Dispense and read devices
• Important for measuring of activity after addition of liquide
 Generell error sources
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Crosstalk
Volume effects
• Luft bobbles
• cracks
• Light scattering
Robotics
Robotics
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Traditional workstation
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simple functions
-> Plate storage
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• Specific and effectiv
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• from simple plate storage
• to advanced systemes
Plate reader or liquide
handling
Plates moved manual
-> Robot treatment of samples
From 1 pipette to 4 og 8
channel pipettes
More flexible
Developed from advanced
systems
• Liquide handling systems
• full automated
”Hotels”
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carousel storage
Automatic stregkode
Temperatur/fugtigheds
kontrol
”Plate Stacker”
• Lodrette stabler
• first in – first out system
-> Plate transfer
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Simple – limited to 2 axes
”high-tech” – faster, higher
flexibility.
Robotics
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full integrated robotic systems
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collection of different workstations
Robotic arm looks for moved plates between workstations
Controlled by Software
”turn-key” systemer
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Ready for use -> expensive
delivered with verified protocols and software
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The Genesis Robotic Sample Processors (RSP) Pro Team 150 combines the highest standards of liquid
handling capabilities with powerful new abilities. Transport of reaction containers by the optional
Robotic Manipulator arm (RoMa) allows integration with plate washers, readers and a wide range of
other sophisticated devices. Genesis Series software and hardware is designed for easy integration
with other automated systems, building towards a fully automated laboratory.
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The CyBi®-Lumax flash is designed to fit easily into robotic systems for fully automated and unattended
performance of flash luminescence assays. For example the reader provides robot-accessible plate
loading, software interfaces for remote control and a remarkably small footprint (57 x 62 cm). The reservoirs
for cells and reagents as well as stirring devices for cell suspensions can be chosen according to the application
needs, since they are placed outside of the reader, which also simplifies refilling or exchange of reservoirs
without the need to open the device.
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TAP SelecT. Schematic diagram and picture of automated cell culture system (TAP
SelecT). It has incubator for 182 flasks T-175 cm2 Incubator for 420 microtiter plates;
cell counter Cedex; carousel for empty flasks; noncontact multiformat cell dispenser TAP
MFCD dispenser; pipette holder; and articulating robotic arm to move flasks and pipettor
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