Introduction to V&P Scientific Pin Tools and Accessories for Tecan Workstations Patricia Koutz Director of R&D

Advantages of V&P Pin Tools Delivery from 2 nl to 5 ul. Nanoliter delivery with CVs averaging ~5% fluorescein. Convert a retired 96 robot to 384 or 1536 pin tool station. Accommodates 1536 format easily. Nanoliter delivery facilitates assay miniaturization. Pin tool assay procedure shortens cycle time by half.

Advantages of V&P Pin Tools Eliminates dilution step in screening assays to save time, reagents and disposable costs. (10-50nL direct into assay plate) No disposable tip costs – pays for tool by second run. Method development is quick. Pin tools mount to 45 commercial robot systems. Pin tools are useful in many applications.

Tecan Floating Pin Tool – MCA/TeMO

Tecan Floating Pin Tool – RoMA

What Makes a Pin Tool? The Pins The Fixture- 2 float plates The Mounting Plate

Slot pin with liquid in slot V&P Pins Large selection of pin diameters, slots, lengths, and coatings to accommodate many applications. Slot width is typically less than 1/3 the diameter of the pin. Solid pin with hanging drop Slot pin with liquid in slot 7 7

V&P Pin Diameters

Tube & E-Clip Style Pins

E-Clip Style Pins Common Uses Larger Volume Transfers (500nl-5ul) Cell Transfers (Yeast, E. coli, etc.): Liquid-to-liquid (Solid or slotted pins) Liquid-to-membrane Liquid-to-agar (Solid pins only) Agar-to-agar 1.58mm 4.77mm 2.36mm 3.18mm

FP6 Slot Pins 1.58mm diameter

Tube-Style Pins Common Uses Very Small Volume Transfers (2-500nl) Compound Transfers

FP1 Slot Pins

FP3 Slot Pins

Misc. Info Liquid transfers with slotted pins Slots fill by capillary action Slots do not need to be submerged to fill Slots MUST be submerged to transfer liquid The slot of the FP6S5 pin (5ul volume) is 6mm deep. Watch liquid displacement in wells of microplate as pins enter the wells.

V&P Floating Pin Tool Fixture

V&P Mono Pin Tool Fixture Mono Pin Tool on MWG RoboAmp 17 17

V&P Rotational Mounting Plate for Tecan MCA/TeMO Pin Tools

V&P Mounting Plate for Tecan RoMA Pin Tools

Guide for Selecting the Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck.

Guide for Selecting the Right Pin Tool Robot system. V&P RoMA Mounting Plate (BMPROMA) V&P standard MCA/TeMO mounting plate (BMPTECAN) V&P rotational MCA/TeMO mounting plate (BMPTECANR) Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck.

Tecan TeMO Pin Tool (Yale)

Tecan RoMA Pin Tool (U of Kentucky)

V&P Docking Station for Tecan Pin Tools 24 24

V&P Docking Station for Tecan Pin Tools 25 25

Guide for Selecting the Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck.

Factors Affecting Pin Delivery Volume Pin diameter. Volume of slot in pin. Surface tension of the liquid being transferred. Surface tension of the pin. – how clean? Uneven transfers due to the sides and slot volume variation Depth to which the pin is submerged in source plate. SPEED OF REMOVAL OF PIN FROM SOURCE LIQUID. CLEAN PINS THOROUGHLY!!!! Don’t use polystyrene source plates.

Speed of removal of pins Pull pins out SLOW from source plates Excess liquid will wick off and LESS volume will be transferred Pull pins out FAST Excess liquid does not wick off and HIGHER volumes will be transferred INCREASE SPEED = INCREASE VOLUME

Speed of removal of pins During Customer Set Up: Customers set up a standard curve and mock fluorescein assay at various withdrawal speeds to determine the appropriate speed for the volume desired. CV’s generally do not change with speed.

V&P Scientific Nanoliter Pins DIAMETER SHAPE DELIVERY VOLUME RANGE1,2 FP9 0.229 mm Solid 4 - 10 nl FP1 0.457 mm 19 - 54 nl FP1S10 10 nl Slot 29 - 65 nl FP1S50 50 nl Slot 62 - 95 nl FP3 0.787 mm 85 - 230 nl FP3S100 100 nl Slot 178 - 312 nl FP3S200 200 nl Slot 284 - 401nl FP3S500 500 nl Slot 505 - 583 nl 1 Volume range determined by speed of withdrawal from source plate 2 Change in speed 7-fold, liquid transferred in DMSO

What happens exactly? Pin tools are a TRANSFER tool, not a dispensing tool. Example: If you transfer 5 ul from the source plate to 45 ul in the destination plate with a pin tool, you will have 50 ul of a 1:10 dilution in the destination plate…while the pins are in the wells. When the pins are withdrawn from the wells, they will remove 5 ul of the 1:10 dilution, leaving 45 ul final volume in the destination plate. Solid pins transfer liquid on the sides of the pins and larger hanging droplet Slotted pins transfer liquid on the sides, in the slot and hanging droplet

What happens exactly? Pre-wet pins in source plate Move pins up and down through the meniscus, 3X (completely out of the liquid). If the pins hit a air bubble going into well, this mixing will displace it. Similar to pre wetting pipetting tips. Post-transfer mix in destination plate Move pins up and down through the meniscus, 3X. Pins act as piston moving up and down to mix. Just letting pins sit in liquid doesn’t help increase amount of volume transfer.

Last Withdrawal Speed The LAST mix withdrawal of the pins from the source plate is the most important. This speed is determined from the mock fluorescein assay run during set up. Use the same speed and number of cycles in source and destination plates so the transfer amounts will be the same. The first two up and down mix movements can be faster.

Guide for Selecting the Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Non-specific binding to stainless steel. “Z” clearance on robot deck. Solid pins versus slotted pins. Don’t use polystyrene source plates

Affect of Variable Source Plate Volumes on Pin Transfer Volume

Guide for Selecting the Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Cost, CV’s Non-specific binding to stainless steel. “Z” clearance on robot deck. Slot pins are less affected by different levels of liquid in source plate (cherry-picked wells) Entire slot must be submerged for complete transfer.

Hydrophobic/Lipophobic Coating Prevents or reduces non-specific binding. Prevents or reduces lipid and lipoprotein binding. Prevents liquid “creep” up the pin. Increases size of hanging drop. Decreases volume on side of pin. Coating can be applied to all V&P stainless steel pins. At least $2 more vs. uncoated pin. Whole pin coated including slot.

Guide for Selecting the Right Pin Tool Robot system. Volume to be transferred. Volume in source plate. Solid pins versus slotted pins. Non-specific binding to stainless steel. “Z” clearance on robot deck. Long pins versus short pins

Tube-Style Exposed Pin Length

Questions To Ask Customers What is the pin tool format? 96, 384 or 1536 pins? What are the mother and daughter plate formats? 96, 384 or 1536? Standard microplates or deep well plates? What is the typical volume in the mother and daughter plates? For example: Average of 50ul liquid in the 384 well mother plate Average of 200ul liquid in the 96 well daughter plate or agar surface in Nunc Omni Tray for daughter plate. 40

Questions To Ask Customers (Cont.) What volume do you need to transfer between the mother and daughter plate? What type of sample is being transferred? For example: Transferring compound libraries in 100% DMSO from mother plate to aqueous cell-based assays in daughter plates. Transferring bacterial or yeast cells in culture media from mother plate to agar surface in daughter plate. Will the pin tool be attached to the instrument head or be picked up with the gripper arm? 41

Pin Tool Data

Are your pins clean?

Pin Validation Assay (Fluorescein Transfer) Materials V&P Scientific pin tools. CyBi™-Well 96 Liquid Handling Robot. Fluorescein Isothiocyanate (FITC), Isomer 1. Sigma P/N F7250. 100% DMSO. Sigma P/N D2650. 100 mM Tris, pH 8.0. Sigma P/N T3038. Microfluor 96-Well Microtiter Plates, Dynex Technologies P/N 7805. PerkinElmer/Wallac VICTOR2 Multilabel Reader.

Pin Validation Assay Methods Dip pin tool into source plate (3X) Pins removed from source plate at controlled speeds Dip pin tool into assay plate (3X) Wash pins Repeat Withdrawal speeds: 0.78-5.76 cm/sec

Pin Tool Maintenance Daily cleaning – Before and after assay. Cleaning in-between sample transfers. Heavy duty cleaning. Frequently used pin tools - clean once a week Infrequently used pin tools - clean once a month

VP 540DB Blotting Station

VP 110 Pin Cleaning Solution Available in 30ml bottles of 5X concentrated solution. Dilute 1:5 in distilled water. Dilution can be reused up to two months if stored in sealed container.

Cleaning Pins In-Between Sample Transfers (cont.) Thorough washing uses 7 deck positions total 96 & 384 pin tools can be blotted in offset positions in VP 540DB Blotting Station. ~2.25-4.5mm offset. Blot in same position on Blotting Station for each wash station. Watch how high the pins are pushed up into the float plate during blotting. If excess liquid is not blotted from slots, the pins may take 60-90 sec to dry unless a fan or vacuum system is used.

Cleaning Pins In-Between Sample Transfers (cont.) Link to Technical Note for liquid sample transfer with robot pin tools and cleaning robot pin tools: http://www.vp-scientific.com/Technotes/135.pdf

Pin Tool Accessories

Converting Manual to Robotic Pin Tool Tube-style (and some E-Clip-style) Manual V&P Pin Tools can be converted to Tecan-compatible robotic Pin Tool by removal of the Basic Guide Pin Plate (BGP). 53

Tecan Pin Tool Docking Station VP 550A Docking Station and TeMO Adaptor Plate

TeMO Picking Up Pin Tool Movie TeMO Picking Up Pin Tool from Docking Station 55

Movie TeMO Setting Pin Tool into Docking Station

Spillway Wash Reservoirs VP 549EtOH Wash Reservoir VP 549H2O Wash Reservoir

VP 540T Flowing Wash Reservoirs

VP 549PE-EP3 Wash Station NOTE: Squarer corners for 1536 pin tools

VP 546B 384 Fountain Wash Station

VP 546C 384 Fountain Wash Station Constructed with hydrophilic coated aluminum. Large channel diameter (2.25mm diameter) to accommodate most pipet tips in addition to pin tools. Reduced channel inlet orifices (0.35mm) for more uniform flow with all wash liquids.

Disposable Pin Tools

New Pin Tool Developments 3456 Pin Tools. 1st 3456 pin tool currently in development phase.