**3. Considerations for using liquid handling devices**

#### **3.1. Determination of quality assessment descriptors**

**2.6. Solenoid-based devices**

180 Drug Discovery

**2.7. Acoustic devices**

continuous stream (Niles and Coassin 2005).

say-ready plates (Turmel et al. 2010)

**2.8. Microplate washers**

aspirate step per cycle).

Solenoid-based devices are non-contact dispensers that use a positive displacement mecha‐ nism (Bateman et al. 1999). The flow of pressurized liquid is occluded by a solenoid valve, which is actuated by electric current to allow for liquid to pass through the valve. The dis‐ pensed volume is regulated by the fluid pressure, duration of the valve in the open position, solution properties and orifice diameter (Bateman et al. 1999; Niles and Coassin 2005). De‐ pending on the time the valve stays in the open position, the device can eject droplets or a

Acoustic droplet ejection (ADE) is a recent touch-less technology that surges in popularity in recent years. It adopts acoustic energy to propel droplets from various types of solutions with good precision (Ellson et al. 2003; Harris et al. 2008; Rudnicki and Johnston 2009; Shieh et al. 2006). The source plate remains stationary as the transducer and destination plate shuf‐ fle to allow for solution transfer from any well in the source plate to any well in the destina‐ tion plate, the latter one lying in an inverted position (Olechno et al. 2006). This system does not require any additional consumable other than microplates (Olechno et al. 2006), and it speeds up the process by avoiding washing steps and having the capability to prepare as‐

Microplate washers are laboratory instruments designed to automate and expedite assay ap‐ plications, where a washing step is essential. They play an important role in areas such as high-content screening and enzyme-linked immunosorbent assays (ELISA). In 1990, Stobbs developed the first multiple plate washer using readily available materials as a low cost al‐ ternative to the commercially available plate washers of the era (Stobbs 1990). Over the years, fully programmable plate washers have been developed with numerous features. The development of automated plate washers has decreased the time required for laborious washing steps involved in many screening assays and improved reproducibility through standardized plate handling across multiple wash cycles (defined as a single dispense and

The two most critical components of a plate washer are a plate carrier and a manifold con‐ taining a number of fixed stainless steel needle probes for solution dispensing (Fig.1A). This manifold (or a separate manifold depending on the design) aspirates the liquid from the wells after an optional soaking period, leaving a pre-defined residual volume in the wells. A third component is the vacuum/pump assembly, which supplies the necessary pressure dif‐ ferential to drive efficient aspiration. Sunghou Lee first developed an additional vacuum fil‐ tration system integrated with a conventional plate washer to speed up the wash process for applications involving filter plates (Lee 2006). Some plate washers have a built-in magnet or

a vacuum filtration module for handling bead-based assays.

Assessment of instrument performance has become important in order to minimize falsepositive and false-negative rates in high-throughput screening (Taylor et al. 2002). One of the most important figures of merit in evaluating the performance of liquid handlers is accu‐ racy, which is commonly reported as %bias (Rose 1999):

$$\% \text{bias} = 100 \times \left(\frac{V\_M \cdot V\_T}{V\_T}\right) \tag{1}$$

where *VM* is the measured volume and *VT*is the theoretical volume (desired). %bias repre‐ sents the deviation from the desired volume, with a value of 0% indicating no deviation from the true value.

The precision, a measure of reproducibility, is calculated from the mean and standard devia‐ tion (SD) of a set of measurements, and it is reported as percent coefficient of variation (%CV) or relative standard deviation (RSD), as shown in Eq. 2. For most cases, it is adequate to have a bias value below 5% and a CV below 10% (Rose 1999).

$$\% \text{\%} \text{CV} \text{=} 100 \times \frac{\text{SD}}{\text{mean}} \tag{2}$$

There have been several approaches for volume verification, which typically consist of gravimetric or photometric methods. Gravimetric measurements utilize the mass and the density (ρ) of the dispensed solution to determine the volume. It has been used extensively to calibrate and verify the accuracy of liquid dispensers (Bergsdorf et al. 2006; Rhode et al. 2004; Taylor et al. 2002). Typically, the solution is dispensed across a pre-weighed microtiter plate, which is weighed immediately after dispensing to prevent evaporation. %bias can be calculated based on the total weight of the dispensed solution (Wtotal) and the number of dis‐ pensed wells (n):

$$\% \text{bias per well (gravitimetric)} = 100 \times \frac{\left(\frac{W\_{\text{total}}}{n \cdot q}\right)\_{\text{V}\_{\text{T}}}}{\text{V}\_{\text{T}}} \tag{3}$$

several factors that affect the dispensed volumes, including viscosity, density, and tempera‐ ture (McGown and Hafeman 1998). General considerations to prevent undesirable dispens‐

Practical Considerations of Liquid Handling Devices in Drug Discovery

http://dx.doi.org/10.5772/52546

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Tubings tend to stretch after certain period of use, affecting the intended volume to be deliv‐ ered. When not in use, the cartridges should be placed in the "rest" position. In addition, autoclaving the cassettes should be minimized. Dispensing speed and the height of the tips in relation to the plate have to be optimized for the intended reagent, as viscous solutions could miss the targeted well at low dispensing speed and large spacing between the tips and microtiter plate. When working with cells, uneven dispensing can be reduced by increasing the prime volume, constant mixing/stirring the cell suspension source and minimizing cell clumps. Solutions should be dispensed in the center of the well, and plates have to be centri‐ fuged when dispensing low volumes to force droplets at the walls to the bottom of the well. Cassettes should be calibrated regularly as recommended by the supplier and checked for

Protein binding to dispensing components is an important point to consider in the imple‐ mentation of biochemical assays, particularly at low protein concentrations. In some instan‐ ces, enzymes appear to be inactivated over time when dispensing multiple plates using a liquid handler, when in reality the enzymes have been depleted from the solution due to non-specific binding to plastic, silicone and other polymer-based surfaces. This effect is am‐ plified when dispensing sizeable number of plates, as there is larger exposure time of the assay components to the surfaces of reagent reservoirs and dispensing cassette elements. In order to circumvent this problem, blocking reagents can be added to the buffer, plastic sur‐ faces can be coated, or a combination of both. The two major types of blocking reagents are detergents and proteins. It is preferable to use non-ionic detergents such as Tween-20, Triton X-100 or Nonidet-P40. Among the most widely-used protein blockers are bovine serum al‐ bumin (BSA) and casein. Protein blockers are better suited for coating surfaces, as detergents can be easily washed away. Typical working concentrations for detergents range from 0.01 to 0.1%, while protein blockers are used between 0.1 to 3 %. The selection of the appropriate type of blocking reagent and concentration is central to a robust assay. Other less common blocking reagents include polyethylene glycol (PEG), polyvinyl alcohol (PVA) and polyvinyl

pyrrolidone (PVP). Additionally, the use of glass reagent reservoirs is recommended.

Particles can obstruct the flow of a dispensing cassette mainly by blocking the tips. Com‐ plete clogging is fairly easy to recognize, as the lack of fluid coming out of the tips can be visibly noticed. Depending on the degree of obstruction, partial clogging may not be easily perceived by the naked eye, and it is detected only by photometric or gravimetric testing. However, there are certain indications of partial clogging, such as slanted fluid spray or

ing performance and common sources of variations include:

*3.2.1. Uneven dispensing*

tip clogging.

*3.2.3. Clogging*

*3.2.2. Protein binding*

Environmental conditions (e.g. temperature and humidity) have major effects on the relia‐ bility of gravimetric methods, which facilitates evaporation and water uptake for hygro‐ scopic solvents such as dimethyl sulfoxide (DMSO). These factors of variation can be minimized by placing gasketed lids on the microtiter plates immediately following dispense (Taylor et al. 2002).

Absorbance and fluorescence are the most common photometric methods utilized to test the accuracy and precision of the transferred volumes of a liquid handling device. In a study comparing the performance of the two methods on determining the precision in 96-/384-/ 1536-well plates, no significant difference was observed between the 96- and 384-well plates (Petersen and Nguyen 2005). However, to achieve similar results for both fluorescence and absorbance measurements in the 1536-well plate, a centrifugation step was required because of the irregular meniscus shape enhanced by the small well geometry. In another study per‐ formed on liquid handlers with two different mechanisms, absorbance was found to be a more reliable method as long as the pH stability of the dye-buffer solution is maintained (Rhode et al. 2004).

Fluorescence signal is also known to be susceptible to photobleaching, which can be pre‐ vented by shorter excitation times, suitable buffer solutions and adequate concentration of fluorophore (Diaspro et al. 2006; Harris and Mutz 2006). To overcome the problems encoun‐ tered due to signal quenching in DMSO, sulforhodamine 101 was presented as an alterna‐ tive fluorescence dye (Walling 2011). Fluorescein was found to be a suitable probe to use in liquid handling performance quantification as long as the DMSO concentration in the buffer solution does not exceed 1% and the stock solutions are stored in 70-100% DMSO in a dark environment (Harris and Mutz 2006). While photobleaching is not an issue in absorbance, the method is limited by high background levels and lower sensitivity compared to fluores‐ cence (Bradshaw et al. 2007). Based on the physical characteristics of a transferred sample and the material of the consumables, unforeseen interactions may be observed influencing the assay results. Especially, DMSO-containing samples are highly affected by the hydro‐ scopic properties of the solvent, which inflates sample volume (Berg et al. 2001).

#### **3.2. Considerations for using bulk reagent dispensers: Peristaltic-based devices**

A single screening experiment can be costly, requiring valuable compounds and biological reagents. Routine evaluation of liquid handlers, in particularly prior to each run, is a neces‐ sary mean for preventing disastrous outcomes. Simple procedures can be integrated to iden‐ tify problems in a relatively short period of time, which in many instances, can be easily corrected. Routine analysis should be performed with the actual reagents, because there are several factors that affect the dispensed volumes, including viscosity, density, and tempera‐ ture (McGown and Hafeman 1998). General considerations to prevent undesirable dispens‐ ing performance and common sources of variations include:
