In order to define the scope of work, automation requirements of analytical sample pretreatment have to be considered.
However, due to the fact that automating biological sample pretreatment is already established using current existing workstations, these processes have to be regarded first. Subsequently, common analytical processes have to be discussed and compared with the defined requirements of biological applications.
3.1 Common Biological Processes
Using a flowchart, the following Fig. 13 represents the sequence of common biological processes.
Fig 13: Flowchart – Sequence of common biological processes including liquid handling, sample treatment (such as incubation steps, vortex mixing, protein precipitation, and extraction), and detection while using different detection modes (such as absorbance, fluorescence, and luminescence) for the required MTP-assays or off- and online separation (using chromatography and mass spectrometry)
Liquid Handling End Start Detection/Separation Sample Treatment Complexity? Enzymes? Antibodies? YES NO
Buffers are extensively used in the wide range of biochemical assays due to the fact that buffers offer an attractive choice for sustaining biological molecules in their native state [119]. Pipetting buffers supplies stable and nonhazardous pH values (mostly pH = 7.4). Moreover, pipetting media and washing solutions is included. Performing biological applications, liquid handling comprises liquid transfer, liquid aspiration and dispension [120], transfer to waste, and serial dilution [121] while handling small volume ranges (≤ 1ml) [122].
Considering temperature (mostly 37°C), humidity, and atmosphere (CO2 = 5%), incubationsteps are
required if the solutions used in the liquid handling steps contain cell metabolism products, such as enzymes and growth factors and, moreover, bio-molecules, such as proteins, nucleic acids, and nucleotides [123]. Due to their complex matrices, evaporation, sonication, vortex mixing, protein precipitation, and extraction of the samples is necessary. Thereby, sample treatment provides an appropriate form of the analyte for the detection and separation systems.
For the final analysis step assay detection readers are required that are mostly designed for the MTP- format. As assays detection technologies have been advanced by the MTP-format, space-saving and user-friendly multimode (or multi-detection) readers have been developed [124], [125] enabling researchers to perform multiple assay types in one instrument [126]. Nevertheless, for some biological measurements chromatographs and mass spectrometers, such HPLC/MS [127], are needed requiring the injection into the separation/detection system. In contrast, assay detection readers facilitate easy online measurements without any injection step. Common detection modes for MTP-assays are absorbance, fluorescence, and luminescence.
The MTP-development replaced the use of test tubes and allows, therefore, for easy automation. Hence, laboratory robots are mostly designed for especially this format. Due to the nonhazardous conditions and the small volumes that occur during biological applications, MTPs can be used during the whole application process: liquid handling – sample treatment – detection/separation. Automating biological applications, commercially available liquid handlers usually support on-deck integration of additional components, such as washers, heaters, shakers, thermocyclers, incubators, centrifuges, SPE, and magnetic bead separation. Using these additional components, liquid handlers enable liquid handling and sample treatment in one process step.
3.2 Common Analytical Processes ‒ Comparison and Conclusion
Using a flowchart, the following Fig. 14 represents the sequence of common analytical processes.
Fig 14: Flowchart – Sequence of common analytical processes including liquid handling, sample handling (such as weighing and capping), sample treatment (such as derivatization and microwave digestion), detection and separation (such as element- and structure-specific measurements using chromatography and mass spectrometry modes)
YES NO YES NO Liquid Handling Sample Treatment End Solid Matter? Start Sample Handling Converting Analyte? Detection/Separation Sample Handling
Depending on the sample properties, samples have to be dosed by weighing (sample handling) or pipetting (liquid handling) first. For weighing and pipetting the utilization of highly active substances, such as concentrated acids or organic solvents with different viscosities, has to be considered demanding the application of inert materials. Furthermore, comparing to biological applications, analytical sample pretreatment calls for higher volume ranges (in some cases up to liters [65]) that are required in order to dissolve higher quantities of the solid samples with non-homogeneous consistency. Moreover, sample handling, such as individual capping, is required in order to ensure the concentration stability while handling volatile solvents and components, such as hexane, acetone, and ethanol. In detail, crimp caps with septa have to be chosen due to the very good seal that is allowed by this combination [128].
In order to ensure analytical measurements, converting the analyte molecules into an appropriate form for the detection and separation systems is required and can be accomplished by derivatization, separation, and microwave digestion. Performing these sample treatment steps, special conditions, such as non-standard temperatures and pressures, are required. Moreover, the required vessel types have to be chemically inert, temperature and pressure resistant, and capable of handling higher volume ranges. Thus, analytical sample pretreatment calls for a wide range of individual vials and tubes that fulfill the requirements mentioned above.
For the final analysis step, detection and separation systems, such as chromatographs and spectrometers [129], [130], are required in order to allow for the determination of the identity and the concentration of a certain, chosen element. Furthermore, selective structure-specific determination and precise quantification of molecules and mixtures of molecules are ensured using these systems. In contrast, biological assay detection supplies easy cell component analysis while simply reacting (binding, adsorbing, activating signal-pathway) with the target substances (as defined by the chosen assay type). However, the main differences between common biological and analytical processes are depicted in the following table.
Table 3: Comparison of common biological and analytical processes
Process Step Biological Process Analytical Process
Liquid Handling
Handling smaller volume ranges: 1µl up to 1ml Pipetting buffers, media,
and washing solutions Non-hazardous pH values
Depends on samples´ consistency Liquids: equal to biological
processes
For solid matters: 1ml up to 1l Highly active solvents/substances
Sample Handling
No individual capping necessary due to easy liquid handling
Volatile substances
Individual capping necessary to ensure concentration stability
Sample Treatment
Mostly incubation steps Temperature 37°C Atmosphere CO2 = 5%
Derivatization and microwave digestion steps
Wide range of temperatures and pressures
Detection Mostly target based assays Cell component detection
Chromatography, spectrometry Selective analysis of certain
elements, molecules, or mixtures of molecules
Consequently, allowing for the analysis of single elements, small molecules, and mixtures of molecules, the requirements of element- and structure-specific analyses differ significantly from biological applications. Therefore, commercially available automated systems are not suitable for analytical sample pretreatment. Furthermore, existing systems either handle only a few steps (mostly at the end) of a complex analytical scheme or they offer a single solution for a fixed process. Therefore, the purpose of this dissertation is the design, the realization, and evaluation of a flexible, automated system that will be capable of
1. Processing extensive analytical applications while considering the specific process requirements, such as the handling of higher volume ranges and highly active substances, non-standard temperature and pressure conditions, and the selective analysis of single elements, molecules, or mixtures of molecules.
2. Processing upgradeable automation steps and flexible analytical sample pretreatment while dealing with the wide range of required vessels simultaneously in order to provide less cost- and time-consuming steps.