Requirements for an Assay 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
Ion Channels by Atomic Absorption At BioFocus, Cambridge
assembly of proteins circular arrangement of proteins closely packed around water-filled pore large-pore channels permit –passage of ions –more or less indiscriminate What is an ion channel ?
archetypal channel pore –one or two atoms wide –conducts a specific species of ion –sodium or potassium –conveyed through membrane single file –fast –access to the pore is governed by a "gate“ –opened or closed by chemical/electrical signals Temperature mechanical force
Redistribution voltage-dependent dyes FRET-based technology Radioligand binding Automated two-electrode voltage clamp Automated whole-cell patch clamp Planar patch clamp Radiometric ion flux Non-radiometric ion flux Measuring Ion Channels Non-radiometric ion flux Radioactive Cost Low throughput
Automated Assay Ion Channel Screening Cells processed using appropriate automation Supernatants analysed for Ion Content –Single burner system (low throughput) –Multi burner system
Automated Platform: Reader platform initial design SOLAAR S AAS #1 AutoSampler 4 Position #1 SOLAAR S AAS #2 AutoSampler 4 Position #2 SOLAAR S AAS #3 AutoSampler 4 Position #3 SOLAAR S AAS #4 AutoSampler 4 Position #4 Linear Track Robotic arm 80 microplate On-line Storage Operating system e.g. OVERLORD™ Data Processing Activity Base All equipment “off the shelf”
Schematic AA #3 AA #4 AA #2 AA #1 Robot bc stacker
What is the challenge for this assay ? 1.Scheduler has to be flexible to time delays 2.SOLAAR software-no automation interface 3.Four AAs required to achieve throughput 4.Autosampler was not robot friendly 5.Safety 6.Data handling/data quality
1. Scheduler needs to be flexible AA run times not the same Throughput critical Error trapping and recovery a necessity
Scheduler control options Time resolved –Static –Pre-emptive –All decisions are taken before the run starts. i.e. before the “GO” button is pressed Event driven –Real time –Dynamic –All decisions are taken during the time frame of the run, i.e. once the “GO” button is pressed Process Analysis & Automation have both !
Time resolved Advantages: –one or more accurate timings are guaranteed –easy to set up Disadvantages –inflexible –decisions can not be taken during the run –error recovery more difficult
Event Driven Advantages: –flexible –decisions can be taken during the run –error recovery easy Disadvantages –accurate timings are more difficult to set up –two accurate timings in a run are impossible
Scheduling - the Choice real-time OVERLORD Workstation pre-emptive OVERLORD Scheduler either
Real Time Instrument Control Options Sequential Round robin
Control system Main controller & Data processing OVERLORD AA#1 instrument control NetLORD node 1 AA#2 instrument control NetLORD node 2 AA#3 instrument control NetLORD node 3 AA#4 instrument control NetLORD node 4 Multiple port switchSite computer services
Instrument control – Round Robin Load, unload busy, error ? AA #2 node Ready ? Error AA #3 node Ready ? AA #4 node Ready ? Start Run Run AAS Load plate AA #1 node Ready ? Data HandlingCycle Run complete ? Unload plate Busy
2. SOLAAR software Requires automation interface There isn’t one !!!! Use the OVERLORD keystroke API Emulates a user
3. Four AAs required for throughput Use NetLORD Remote OVERLORD nodes Makes control structure easier –Node in 4 states Ready for load Ready for unload Busy Error state NetLORD nodes addressed by IP address Common folders for data transfer Redundancy
5. Safety Hazards –AAS instrument Locked room Air conditioning –Acetylene gas Only run during the day Gas detectors Time out on AAS flames –Robot Hamilton SWAP stops when touched Hamilton SWAP can be recovered
6. Data handling/data quality Barcodes Data file renaming for integrity Data transfer to the server Real time data analysis for quality check
Conclusions Instruments can be automated –Keyboard control –Plate access Unusual assays –Real time scheduler –Round robin method Twister I robots good tools –Need more than basic software OVERLORD can control > 220 instruments And remember …..