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automate.it - Atomic Absorption Spectrometer
Determining Ion Channels
by using Atomic Absorption Spectroscopy |
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Background |
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Process Analysis & Automation were commissioned by Biofocus Ltd. To design and build an automated multi-atomic absorption spectrometer system. Using four Thermo Electron SOLAAR AA spectrometers (with associated Gilson autosamplers), a Hamilton Microlab SWAP robot and a Kendro stacker, a fully automated and reliable workcell was produced. OVERLORD dynamic scheduler (Overlord2) was used control the workcell, with NetLORD used to control the four AA control PCs. We believe that this is the only fully commercially available automated AA system.
The following information is taken from a talk by Dr Adrian Kinkaid of Biofocus Ltd (Cambridge) presented to the European Laboratory Robotics Interest Group at GSK, Stevenage in 2004. Biofocus Ltd offer a fully automated service, and details of the service can be found at their web page www.biofocus.com.
This system integrates equipment from Thermo Elemental, Hamilton, Kendro and Metrologic.
Process Analysis & Automation offer a wide range of standard and user defined automation workcells. For advice on system integration: go to System Integration or Download System Integration application note (LA223).
For a copy of the full Biofocus presentation by Adrian Kinkaid presented at ELRIG entitled "Fully Automated High Throughput Ion Channel Screening", download from the PAA downloads page |
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Requirements for an automated Ion Channel Analysis system |
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- High-throughput
- Low false-positive rate
- Low false-negative rate
- Direct measure of function
- Good correlation with electrophysiology
- Reliability
- Reproducibility
- Amenable to miniaturization
- Low cost
- hERG used as a model channel
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Ion Channel screening technologies available (used for hERG) |
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Fluorescence-based assays
- Membrane potential-sensitive dyes
Radioligand binding assays - [3H] Dofetilide Automated electrophysiology
- Automated two-electrode voltage clamp systems
- Automated whole-cell patch clamp systems
- Planar patch clamp techniques
Rubidium efflux assays
- Cerenkov counting of 86 Rb+
- Atomic absorption spectrometry of 85 Rb+
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Which is the best technique ? |
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Technique |
Throughput |
Information quality |
Cost |
Comments |
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Redistribution of voltage-dependent dye |
High |
Medium |
Low |
Compound Interference |
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FRET-based technology |
High |
Medium/High |
High |
- |
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Radioligand binding |
High |
Low |
Low |
Non-functional/ Radioactive |
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Automated two-electrode voltage clamp |
Low/Medium |
High |
High |
Low efficacy |
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Automated whole-cell |
Low/Medium |
High |
High |
Cell dialysis |
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Planar patch clamp |
Medium/High |
High |
High |
Cell dialysis |
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Radiometric ion flux |
High |
Medium |
Low |
Radioactive |
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Non-radiometric ion flux |
High |
Medium |
Low |
- |
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Chosen technique |
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Non-radiometric ion flux was chosen as the method of choice because of high throughput relative to other techniques, low cost, direct measurement of channel activity and does not require radiometric reagents.
The Thermo Elemental AAS (right) was selected as the most suitable instrument for the process. |
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Sample preparation method |
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Robot loading AA autosampler
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- Cells in 96 well plates
- Add dilute compound and incubate
- Add High K + Buffer and incubate
- Transfer supernatant to deep well block or plate
- Make up to 1ml or 330ul with 0.1% CsCl Solution
- Seal and Store
- Read
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Reading the plates |
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Robot taking plate from storage and passed barcode reader
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The plates are read in batches of four plates.
- Remove the plate seals by hand and place in Kendro stacker
- Take each plate in turn
- Read the barcode
- Place plate in the next available AA spectrometer Autosampler location
- Load up 4 plates
- Start the AA run (each of the 384 wells are sampled, and the Rubidium content determined)
- Plates are returned the stacker
- Results are collated with the plate number and sent to the company server for processing
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