Liquid handling steps

Cell harvesters were developed to capture multiple samples of cells on membrane filters, wash away unincorporated isotopes, and prepare samples for liquid scintillation counting on special equipment developed to process and count multiple samples. Despite miniaturization and improvements in efficiency of this technique, the disadvantages of multiple liquid handling steps and increasing costs for disposal of radioactive waste materials severely limit its usefulness. Although specific applications require measuring DNA synthesis as a marker for cell proliferation, much better choices are available for detecting viable cell number for HTS. 

HCS assays have routinely been formatted in 96- and 384-well plates and reports of 1536-well assays have emerged at conferences. Equipment suited to manipulate these plates and manage the associated liquid handling steps in bulk is absolutely required to achieve any scale over a few 96-well plates at a time. Bulk liquid dispensers such as the WellMate and liquid transfer devices like the Beckman FX are commonly found in most assay development and screening labs and are essential for HCS. Plate washing is usually needed for HCS assays and presents an important step to optimize. As discussed earlier, cell adherence must be maintained and adequate cell washing at... 

However, it has to be kept in mind that the main motivations for the application of assays based on microfluidics and microarrays are (i) their ability to enable experiments with biological samples that are only available in very small quantities (few microliters), and (ii) the reduction of the number of liquid-handling steps compared to the same assay in a well plate. These aspects are further discussed in Section 3. 

As stated in Section 1 of this chapter, the diversity of hqnid samples is handled by confining each sample within the walls of a compartment. Planar arrays of snch sample compartments are realized in the well plates discnssed above. The hqnid-handling process then needs to address each sample of a diverse collection individually, which imposes a limit for further miniaturization. Therefore, successfnl miniaturization concepts will lead to a reduction of the nnmber of liquid-handling steps compared to well-based approaches. Miniaturization is particularly enabling in sitnations where precious biological fluids that are only available in limited qnanti-ties need to be tested against a set of samples. 

Figure 7 Fluorescence image of a sandwich immunoassay that was generated with the microchannel assay system. Capture antibodies were patterned as 11 vertical lines. Subsequently, 17 different biological samples were patterned as horizontal lines. At the intersections, an interaction pattern of 11 X17 = 187 features is thus generated with 11 + 17 = 28 liquid-handling steps...

Instead of the time-consuming and expensive developments of application-specific microfluidic solutions, we propose to base microfluidic developments on a microfluidic platform approach, where a combinable set of liquid handling steps together with a suitable fabrication technology enable the flexible and affordable implementation of biochemical protocols in a market-relevant... 

Conveying this to the microfluidic platform approach, a set of validated microfluidic elements is required, each able to perform a certain basic liquid handling step or unit operation. Such basic unit operations are building blocks of laboratory protocols and comprise fluid transport, fluid metering, fluid mixing, valving, separation or concentration of molecules or particles (see Table 1) and others. Every microfluidic platform should offer an adequate number of microfluidic unit operations that can be easily combined to build application-specific microfluidic systems. 

How this works in practice is detailed as follows. After a compound has been identified for which several hundred to several thousand derivatives would be of value, a synthetic route is chosen that (i) permits linkage to a solid-phase support (ii) utilizes reaction steps that appear possible to optimize to > 90% yield and (iii) affords reagents in each step for which desirable variants can be purchased (or, less optimally, can be made trivially). In the synthesis itself, one of the significant advantages of the microreactor approach becomes evident one can use standard laboratory glassware and equipment to accomplish the library synthesis. There is no need for the automation of liquid-handling steps, and indeed no need for automation at all until rather large libraries are desired (vide supra). 

Automation using a robotic liquid handling system eliminated most of the tedious steps encountered with traditional manual extraction procedures. Automated 96-well SPE and LLE techniques using robotic liquid handlers have been successfully implemented to support high-throughput bioanalysis.5... 

Typical protein precipitation procedures use one volume of plasma plus three to six volumes of acetonitrile or methanol (or a mixture) with the internal standard at an appropriate concentration for the assay. Poison et al.102 reported that protein precipitation using acetonitrile eliminates at least 95% of the proteins after filtration or centrifugation, the supernatant can often be directly injected into the HPLC/MS/MS system. Usually this step is performed using 96-well plates that are ideal for semi-automation of sample preparation. Briem et al.103 reported on a robotic sample preparation system for plasma based on a protein precipitation step and a robotic liquid handling system that increased throughput by a factor of four compared to a manual system. 

The next step is to determine the solubility of the substrate (or its salts) in different solvents. This can also be performed by an automated liquid handling system. Depending upon the solubility of the substrate in water-miscible solvent (alcohols, acetone, tetrahydrofuran, etc.) and water-immiscible solvents (ethyl acetate, methyl-tert-butyl ether, heptane, etc.) the process chemist can identify one or many solvent systems from which the substrate (or its salts) could be ciystallized using the antisolvent addition strategy. 

The siRNA libraries are obtained as a 10 pM stock and we prepare a 1 30 dilution resulting in 333 nM daughter plate concentrations. Although this step can be done manually using multichannel pipets, it can be greatly expedited by the use of a liquid-handling system, such as the Biomek EX Workstation (Beckman Coulter Brea, CA). 

By GLC methods, it is possible to obtain detailed quantitative analyses of saturates up to C9, of mono-olefins up to C7, and of aromatics up to Cio (48). The liquid chromatographic steps cannot handle materials boiling below n-Ci2. Therefore, there is a gap in the analysis of the distillate fractions on which a detailed analysis cannot be readily, routinely, and inexpensively obtained. This gap includes all the Cg-Ci2 saturates, all the Cn-Ci2 aromatics, and all the heterocompounds and olefins that are present in this fraction. 

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