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Precision Testing for the Sciclone Advanced Liquid Handler Z8 Option

Jeff Cunningham and Lynn Jordan
Caliper Life Sciences, Inc., Applications Laboratory, Hopkinton, Massachusetts, USA

Introduction

The Z8 liquid handling device is an optional accessory to the Sciclone Advanced Liquid Handler (ALH). This device is designed to permit individually and independently controlled access to single wells or columns of 96 and 384 well microplates for purposes of liquid transfer (tertiary dispensing). It can be configured to use fixed cannulas (1-50μL) or disposable tips (1-100μL) to facilitate standard hit-picking or re-formatting protocols. Here, we discuss the optimization of liquid transfer (1, 2, 5 and 10μL) techniques in terms of precision. Moreover, we extend the capabilities of the Z8 by performing continuous low volume (1-10μL) bulk dispensing across the columns of a 96 well plate (secondary dispensing) using fixed cannulas.

Materials and Methods

Instrumentation

All testing was performed using a Sciclone ALH with the Z8 option. Secondary dispensing is achieved using a Bulk Reagent Dispenser (Sciclone option) which facilitates the continuous delivery of water across the columns of a plate. Absorbance was measured at 450nm using a Wallac Victor2 Multilabel Counter (PerkinElmer).

Reagents

Tartrazine (SIGMA) solutions are prepared with deionized H2O and degassed with Helium at 0.7, 1.4, 5.0, and 6.0g/L) and dispensed at 10, 5, 2 and 1μL respectively.

Device Purging

The Z8 device is first purged to facilitate the elimination of compressible materials or gasses as follows:

  • Prime the lines of the Z8 until the system fluid reaches the lines just past the syringes.
  • Aspirate 100μL from source.
  • Manually remove trapped air in the syringe barrels by first loosening the plunger mounting screws then pushing up on each plunger 3-4 times quickly.
  • Re-prime the lines until liquid flows from the ends of the tip adapters or fixed cannulas.

As a result of the purging, the system fluid (in this case degassed and deionized water) forms a continuous path from its source, through pumps, lines and syringes to individual fixed cannulas or tip adapters.

Disposable Tip Precision Determination (1, 2, 5μL tertiary dispensing):

All testing was performed using the deck layout (Fig.1) and a protocol to execute the following:

  • Deliver 100μL of water into each well of a NUNC (Denmark) 96 well, clear bottom, untreated plate using the Bulk Dispenser.
  • Attach 100μL Automation Certified tips to the Z8 tip-adapters.
  • "Pre-wet" the tips by triturating 50μL of tartrazine solution.
  • Aspirate a 5μL air gap followed by 1, 2 or 5μL of tartrazine.
  • Dispense total content (tartrazine and air gap) into the first column of the 96 well plate and repeat the process for all columns.
  • Dispense 100μL of water into each well using the Bulk Dispenser.
  • Acquire and export absorbance data to ExcelTM for CV calculation.
  • Repeat for each of 5 plates at appropriate tartrazine concentrations.

Fixed Cannula Precision Determination (1, 2, 5 and 10μL tertiary dispensing):

All testing performed using the deck layout (Fig.2) and a protocol to execute the following:

  • Deliver 100μL of water into each well of a 96 well plate using the Bulk Dispenser.
  • Aspirate 10μL of air followed by tartrazine (1, 2, 5 or 10μL) and a final 5μL air gap.
  • Dispense total content (tartrazine and both air gaps) into first column of the 96 well plate.
  • "Quick wash" cannulas by priming 100μL into a waste reservoir twice then triturate 100μL of clean water three times.
  • Repeat previous four steps for all 12 columns.
  • Deliver 100μL of water into each well of a 96 well plate using the Bulk Dispenser.
  • "Thoroughly wash" cannulas by priming 100μL into a waste reservoir four times then triturate 100μL of clean water three times.
  • Acquire and export absorbance data to Excel for CV calculation.
  • Repeat for each of five plates at appropriate tartrazine concentrations

Fixed Cannula Precision Determination (1, 2 and 5μL secondary dispensing):
All testing performed using the deck layout (Fig.3) and a protocol to execute the following:

  • Deliver 100μL of water into each well of a 96 well plate using the Bulk Dispenser.
  • Aspirate air gap (10μL) followed by tartrazine solution (25μL) for a total of 35μL.
  • Dispense appropriate volume of tartrazine solution (1, 2 or 5μL) once into a waste reservoir then into each well of the test plate.
  • Deliver water (100μL) into each well with the Bulk Dispenser.
  • Acquire and export absorbance data to Excel for CV determination.< br>
  • Repeat for each of five plates at appropriate tartrazine concentrations.
  • Prime cannulas four times with water (100μL) into a waste reservoir.
  • Triturate cannulas for three cycles with water (100μL).

Results

Absorbance was measured and the data exported to ExcelTM for CV determination for five plates at each volume tested. The results of the dispense data for disposable tips (Table 1), fixed cannula secondary dispensing (Table 2) and fixed cannula tertiary dispense data (Table 3) are shown below. The overall CV's that comprise every well of a 96 well plate are shown for each volume tested. The mean %CV is caculated in each table.

Discussion

In this study, we explore the capabilities of the Sciclone ALH Z8 option using both disposable tips and fixed cannulas. We demonstrate its utility for tertiary dispensing (e.g., hit-picking), where small volumes of substance are transferred from single wells and dispensed into new destination wells. Moreover, we show that the functionality of the Z8 can be extended to perform low volume (1-10μL) secondary dispensing. This is noteworthy since the Bulk Dispenser option on the ALH is rated to dispense volumes of 10μL or greater. (see, Cunningham J and Jordan L "Sciclone ALH Bulk Dispense Precision Testing", Application Note-AP5, Zymark Corporation, 2001)

The precision results achieved in this study surpass the published factory specifications for disposable tips and fixed cannulas (CV <= 10% and 5%, respectively), at 1μL dispense volumes. While this is likely a result of good pipetting technique such as proper tip attachment and optimal aspirating and dispensing heights, we have found that purging the system of all air is equally important for proper Z8 functionality.


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