Automation of peptide desalting for proteomic liquid chromatography - tandem mass spectrometry by centrifugal microfluidics.

For large-scale analysis of complex protein mixtures, liquid chromatography - tandem mass spectrometry (LC-MS/MS) has been proven to be one of the most versatile tools due to its high sensitivity and ability to both identify and quantify thousands of proteins in a single measurement. Sample preparation typically comprises site-specific cleavage of proteins into peptides, followed by desalting and concomitant peptide enrichment, which is commonly performed by solid phase extraction. Desalting workflows may include multiple liquid handling steps and are thus error prone and labour intensive. To improve the reproducibility of sample preparation for low amounts of protein, we present a centrifugal microfluidic disk that automates all liquid handling steps required for peptide desalting by solid phase extraction (DesaltingDisk). Microfluidic implementation was enabled by a novel centrifugal microfluidic dosing on demand structure that enabled mapping multiple washing steps onto a microfluidic disk. Evaluation of the microfluidic disk was performed by LC-MS/MS analysis of tryptic HEK-293 eukaryotic cell peptide mixtures desalted either using the microfluidic disk or a manual workflow. A comparable number of peptides were identified in the disk and manual set with 19 775 and 20 212 identifications, respectively. For a core set of 10 444 peptides that could be quantified in all injections, intensity coefficients of variation were calculated based on label-free quantitation intensities. The disk set featured smaller variability with a median CV of 9.3% compared to the median CV of 12.6% for the manual approach. Intensity CVs on protein level were lowered from 5.8% to 4.2% when using the LabDisk. Interday reproducibility for both workflows was assessed by LC-SRM/MS analysis of samples that were spiked with 11 synthetic peptides of varying hydrophobicity. Except for the most hydrophilic and hydrophobic peptides, the average CV was lowered to 3.6% for the samples processed with the disk compared to 7.2% for the manual workflow. The presented centrifugal microfluidic DesaltingDisk demonstrates the potential to improve reproducibility in the sample preparation workflow for proteomic mass spectrometry, especially for application with limited amount of sample material.

Tryptic digestion of human serum for proteomic mass spectrometry automated by centrifugal microfluidics.

Mass spectrometry has become an important analytical tool for protein research studies to identify, characterise and quantify proteins with unmatched sensitivity in a highly parallel manner. When transferred into clinical routine, the cumbersome and error-prone sample preparation workflows present a major bottleneck. In this work, we demonstrate tryptic digestion of human serum that is fully automated by centrifugal microfluidics. The automated workflow comprises denaturation, digestion and acidification. The input sample volume is 1.3 µl only. A triplicate of human serum was digested with the developed microfluidic chip as well as with a manual reference workflow on three consecutive days to assess the performance of our system. After desalting and liquid chromatography tandem mass spectrometry, a total of 604 proteins were identified in the samples digested with the microfluidic chip and 602 proteins with the reference workflow. Protein quantitation was performed using the Hi3 method, yielding a 7.6% lower median intensity CV for automatically digested samples compared to samples digested with the reference workflow. Additionally, 17% more proteins were quantitated with less than 30% CV in the samples from the microfluidic chip, compared to the manual control samples. This improvement can be attributed to the accurate liquid metering with all volume CVs below 1.5% on the microfluidic chip. The presented automation solution is attractive for laboratories in need of robust automation of sample preparation from small volumes as well as for labs with a low or medium throughput that does not allow for large investments in robotic systems.

Library preparation for next generation sequencing: A review of automation strategies.

Next generation sequencing is in the process of evolving from a technology used for research purposes to one which is applied in clinical diagnostics. Recently introduced high throughput and benchtop instruments offer fully automated sequencing runs at a lower cost per base and faster assay times. In turn, the complex and cumbersome library preparation, starting with isolated nucleic acids and resulting in amplified and barcoded DNA with sequencing adapters, has been identified as a significant bottleneck. Library preparation protocols usually consist of a multistep process and require costly reagents and substantial hands-on-time. Considerable emphasis will need to be placed on standardisation to ensure robustness and reproducibility. This review presents an overview of the current state of automation of library preparation for next generation sequencing. Major challenges associated with library preparation are outlined and different automation strategies are classified according to their functional principle. Pipetting workstations allow high-throughput processing yet offer limited flexibility, whereas microfluidic solutions offer great potential due to miniaturisation and decreased investment costs. For the emerging field of single cell transcriptomics for example, microfluidics enable singularisation of tens of thousands of cells in nanolitre droplets and barcoding of the RNA to assign each nucleic acid sequence to its cell of origin. Finally, two applications, the characterisation of bacterial pathogens and the sequencing within human immunogenetics, are outlined and benefits of automation are discussed.

Review on pneumatic operations in centrifugal microfluidics.

Centrifugal microfluidics allows for miniaturization, automation and parallelization of laboratory workflows. The fact that centrifugal forces are always directed radially outwards has been considered a main drawback for the implementation of complex workflows leading to the requirement of additional actuation forces for pumping, valving and switching. In this work, we review and discuss the combination of centrifugal with pneumatic forces which enables transport of even complex liquids in any direction on centrifugal systems, provides actuation for valving and switching, offers alternatives for mixing and enables accurate and precise metering and aliquoting. In addition, pneumatics can be employed for timing to carry out any of the above listed unit operations in a sequential and cascaded manner. Firstly, different methods to generate pneumatic pressures are discussed. Then, unit operations and applications that employ pneumatics are reviewed. Finally, a tutorial section discusses two examples to provide insight into the design process. The first tutorial explains a comparatively simple implementation of a pneumatic siphon valve and provides a workflow to derive optimum design parameters. The second tutorial discusses cascaded pneumatic operations consisting of temperature change rate actuated valving and subsequent pneumatic pumping. In conclusion, combining pneumatic actuation with centrifugal microfluidics allows for the design of robust fluidic networks with simple fluidic structures that are implemented in a monolithic fashion. No coatings are required and the overall demands on manufacturing are comparatively low. We see the combination of centrifugal forces with pneumatic actuation as a key enabling technology to facilitate compact and robust automation of biochemical analysis.

Temperature change rate actuated bubble mixing for homogeneous rehydration of dry pre-stored reagents in centrifugal microfluidics.

In centrifugal microfluidics, dead volumes in valves downstream of mixing chambers can hardly be avoided. These dead volumes are excluded from mixing processes and hence cause a concentration gradient. Here we present a new bubble mixing concept which avoids such dead volumes. The mixing concept employs heating to create a temperature change rate (TCR) induced overpressure in the air volume downstream of mixing chambers. The main feature is an air vent with a high fluidic resistance, representing a low pass filter with respect to pressure changes. Fast temperature increase causes rapid pressure increase in downstream structures pushing the liquid from downstream channels into the mixing chamber. As air further penetrates into the mixing chamber, bubbles form, ascend due to buoyancy and mix the liquid. Slow temperature/pressure changes equilibrate through the high fluidic resistance air vent enabling sequential heating/cooling cycles to repeat the mixing process. After mixing, a complete transfer of the reaction volume into the downstream fluidic structure is possible by a rapid cooling step triggering TCR actuated valving. The new mixing concept is applied to rehydrate reagents for loop-mediated isothermal amplification (LAMP). After mixing, the reaction mix is aliquoted into several reaction chambers for geometric multiplexing. As a measure for mixing efficiency, the mean coefficient of variation (C[combining macron]V[combining macron], n = 4 LabDisks) of the time to positivity (tp) of the LAMP reactions (n = 11 replicates per LabDisk) is taken. The C[combining macron]V[combining macron] of the tp is reduced from 18.5% (when using standard shake mode mixing) to 3.3% (when applying TCR actuated bubble mixing). The bubble mixer has been implemented in a monolithic fashion without the need for any additional actuation besides rotation and temperature control, which are needed anyhow for the assay workflow.

Network simulation-based optimization of centrifugo-pneumatic blood plasma separation.

Automated and robust separation of 14 µl of plasma from 40 µl of whole blood at a purity of 99.81% ± 0.11% within 43 s is demonstrated for the hematocrit range of 20%-60% in a centrifugal microfluidic polymer disk. At high rotational frequency, red blood cells (RBCs) within whole blood are concentrated in a radial outer RBC collection chamber. Simultaneously, plasma is concentrated in a radial inner pneumatic chamber, where a defined air volume is enclosed and compressed. Subsequent reduction of the rotational frequency to not lower than 25 Hz enables rapid transfer of supernatant plasma into a plasma collection chamber, with highly suppressed resuspension of red blood cells. Disk design and the rotational protocol are optimized to make the process fast, robust, and insusceptible for undesired cell resuspension. Numerical network simulation with lumped model elements is used to predict and optimize the fluidic characteristics. Lysis of the remaining red blood cells in the purified plasma, followed by measurement of the hemoglobin concentration, was used to determine plasma purity. Due to the pneumatic actuation, no surface treatment of the fluidic cartridge or any additional external means are required, offering the possibility for low-cost mass fabrication technologies, such as injection molding or thermoforming.

C-reactive protein and interleukin 6 microfluidic immunoassays with on-chip pre-stored reagents and centrifugo-pneumatic liquid control.

We present a fully automated centrifugal microfluidic method for particle based protein immunoassays. Stick-pack technology is employed for pre-storage and release of liquid reagents. Quantitative layout of centrifugo-pneumatic particle handling, including timed valving, switching and pumping is assisted by network simulations. The automation is exclusively controlled by the spinning frequency and does not require any additional means. New centrifugal microfluidic process chains are developed in order to sequentially supply wash buffer based on frequency dependent stick-pack opening and pneumatic pumping to perform two washing steps from one stored wash buffer; pre-store and re-suspend functionalized microparticles on a disk; and switch between the path of the waste fluid and the path of the substrate reaction product with 100% efficiency. The automated immunoassay concept is composed of on demand ligand binding, two washing steps, the substrate reaction, timed separation of the reaction products, and termination of the substrate reaction. We demonstrated separation of particles from three different liquids with particle loss below 4% and residual liquid remaining within particles below 3%. The automated immunoassay concept was demonstrated by means of detecting C-reactive protein (CRP) in the range of 1-81 ng ml-1 and interleukin 6 (IL-6) in the range of 64-13 500 pg ml-1. The limit of detection and quantification were 1.0 ng ml-1 and 2.1 ng ml-1 for CRP and 64 pg ml-1 and 205 pg ml-1 for IL-6, respectively.

Robust temperature change rate actuated valving and switching for highly integrated centrifugal microfluidics.

We present new unit operations for valving and switching in centrifugal microfluidics that are actuated by a temperature change rate (TCR) and controlled by the rotational frequency. Implementation is realized simply by introducing a comparatively large fluidic resistance to an air vent of a fluidic structure downstream of a siphon channel. During temperature decrease at a given TCR, the air pressure inside the downstream structure decreases and the fluidic resistance of the air vent slows down air pressure compensation allowing a thermally induced underpressure to build up temporarily. Thereby the rate of temperature change determines the time course of the underpressure for a given geometry. The thermally induced underpressure pulls the liquid against a centrifugal counterpressure above a siphon crest, which triggers the valve or switch. The centrifugal counterpressure (adjusted by rotation) serves as an independent control parameter to allow or prevent valving or switching at any TCR. The unit operations are thus compatible with any temperature or centrifugation protocol prior to valving or switching. In contrast to existing methods, this compatibility is achieved at no additional costs: neither additional fabrication steps nor additional disk space or external means are required besides global temperature control, which is needed for the assay. For the layout, an analytical model is provided and verified. The TCR actuated unit operations are demonstrated, first, by a stand-alone switch that routes the liquid to either one of the two collection chambers (n = 6) and, second, by studying the robustness of TCR actuated valving within a microfluidic cartridge for highly integrated nucleic acid testing. Valving could safely be prevented during PCR by compensating the thermally induced underpressure of 3.52 kPa with a centrifugal counterpressure at a rotational frequency of 30 Hz with a minimum safety range to valving of 2.03 kPa. Subsequently, a thermally induced underpressure of 2.55 kPa was utilized for robust siphon valving at 3 Hz with a minimum safety range of 2.32 kPa.

System-level network simulation for robust centrifugal-microfluidic lab-on-a-chip systems.

Centrifugal microfluidics shows a clear trend towards a higher degree of integration and parallelization. This trend leads to an increase in the number and density of integrated microfluidic unit operations. The fact that all unit operations are processed by the same common spin protocol turns higher integration into higher complexity. To allow for efficient development anyhow, we introduce advanced lumped models for network simulations in centrifugal microfluidics. These models consider the interplay of centrifugal and Euler pressures, viscous dissipation, capillary pressures and pneumatic pressures. The simulations are fast and simple to set up and allow for the precise prediction of flow rates as well as switching and valving events. During development, channel and chamber geometry variations due to manufacturing tolerances can be taken into account as well as pipetting errors, variations of contact angles, compliant chamber walls and temperature variations in the processing device. As an example of considering these parameters during development, we demonstrate simulation based robustness analysis for pneumatic siphon valving in centrifugal microfluidics. Subsequently, the influence of liquid properties on pumping and valving is studied for four liquids relevant for biochemical analysis, namely, water (large surface tension), blood plasma (large contact angle hysteresis), ethanol/water (highly wetting) and glycerine/water (highly viscous). In a second example, we derive a spin protocol to attain a constant flow rate under varying pressure conditions. Both examples show excellent agreement with experimental validations.

Rigorous buoyancy driven bubble mixing for centrifugal microfluidics.

We present batch-mode mixing for centrifugal microfluidics operated at fixed rotational frequency. Gas is generated by the disk integrated decomposition of hydrogen peroxide (H2O2) to liquid water (H2O) and gaseous oxygen (O2) and inserted into a mixing chamber. There, bubbles are formed that ascent through the liquid in the artificial gravity field and lead to drag flow. Additionaly, strong buoyancy causes deformation and rupture of the gas bubbles and induces strong mixing flows in the liquids. Buoyancy driven bubble mixing is quantitatively compared to shake mode mixing, mixing by reciprocation and vortex mixing. To determine mixing efficiencies in a meaningful way, the different mixers are employed for mixing of a lysis reagent and human whole blood. Subsequently, DNA is extracted from the lysate and the amount of DNA recovered is taken as a measure for mixing efficiency. Relative to standard vortex mixing, DNA extraction based on buoyancy driven bubble mixing resulted in yields of 92 ± 8% (100 s mixing time) and 100 ± 8% (600 s) at 130g centrifugal acceleration. Shake mode mixing yields 96 ± 11% and is thus equal to buoyancy driven bubble mixing. An advantage of buoyancy driven bubble mixing is that it can be operated at fixed rotational frequency, however. The additional costs of implementing buoyancy driven bubble mixing are low since both the activation liquid and the catalyst are very low cost and no external means are required in the processing device. Furthermore, buoyancy driven bubble mixing can easily be integrated in a monolithic manner and is compatible to scalable manufacturing technologies such as injection moulding or thermoforming. We consider buoyancy driven bubble mixing an excellent alternative to shake mode mixing, in particular if the processing device is not capable of providing fast changes of rotational frequency or if the low average rotational frequency is challenging for the other integrated fluidic operations.

Centrifugo-pneumatic sedimentation, re-suspension and transport of microparticles.

Microparticles are widely used as solid phase for affinity-based separation. Here, we introduce a new method for automated handling of microparticles in centrifugal microfluidics that is not restricted by the particle size and requires neither auxiliary means such as magnets nor coating of microfluidic structures. All steps are initiated and controlled by the speed of rotation only. It is based on storage and "on demand" release of pneumatic energy within tunable time frames: a slow release of the pneumatic energy triggers a first fluidic path through which the supernatant above the sedimented particles is removed. An abrupt release triggers a second path which allows for liquid routing and transport of the re-suspended particles. Re-suspension of particles is thereby achieved by quickly changing the speed of rotation. We demonstrate the exchange of the particle carrier medium with a supernatant removal efficiency of more than 99.5% and a particle loss below 4%. Re-suspension and subsequent transport of suspended particles show a particle loss below 7%. The method targets the automation of particle-based assays e.g. DNA extractions and immunoassays. It is compatible with monolithic integration and suitable for mass production technologies e.g. thermoforming or injection moulding.

Rapid and fully automated bacterial pathogen detection on a centrifugal-microfluidic LabDisk using highly sensitive nested PCR with integrated sample preparation.

Diagnosis of infectious diseases suffers from long turnaround times for gold standard culture-based identification of bacterial pathogens, therefore impeding timely specific antimicrobial treatment based on laboratory evidence. Rapid molecular diagnostics-based technologies enable detection of microorganisms within hours however cumbersome workflows and complex equipment still prevent their widespread use in the routine clinical microbiology setting. We developed a centrifugal-microfluidic "LabDisk" system for rapid and highly-sensitive pathogen detection on a point-of-care analyser. The unit-use LabDisk with pre-stored reagents features fully automated and integrated DNA extraction, consensus multiplex PCR pre-amplification and geometrically-multiplexed species-specific real-time PCR. Processing merely requires loading of the sample and DNA extraction reagents with minimal hands-on time of approximately 5 min. We demonstrate detection of as few as 3 colony-forming-units (cfu) of Staphylococcus warneri, 200 cfu of Streptococcus agalactiae, 5 cfu of Escherichia coli and 2 cfu of Haemophilus influenzae in a 200 µL serum sample. The turnaround time of the complete analysis from "sample-to-result" was 3 h and 45 min. The LabDisk consequently provides an easy-to-use molecular diagnostic platform for rapid and highly-sensitive detection of bacterial pathogens without requiring major hands-on time and complex laboratory instrumentation.

Centrifugo-pneumatic multi-liquid aliquoting - parallel aliquoting and combination of multiple liquids in centrifugal microfluidics.

The generation of mixtures with precisely metered volumes is essential for reproducible automation of laboratory workflows. Splitting a given liquid into well-defined metered sub-volumes, the so-called aliquoting, has been frequently demonstrated on centrifugal microfluidics. However, so far no solution exists for assays that require simultaneous aliquoting of multiple, different liquids and the subsequent pairwise combination of aliquots with full fluidic separation before combination. Here, we introduce the centrifugo-pneumatic multi-liquid aliquoting designed for parallel aliquoting and pairwise combination of multiple liquids. All pumping and aliquoting steps are based on a combination of centrifugal forces and pneumatic forces. The pneumatic forces are thereby provided intrinsically by centrifugal transport of the assay liquids into dead end chambers to compress the enclosed air. As an example, we demonstrate simultaneous aliquoting of 1.) a common assay reagent into twenty 5 µl aliquots and 2.) five different sample liquids, each into four aliquots of 5 µl. Subsequently, the reagent and sample aliquots are simultaneously transported and combined into twenty collection chambers. All coefficients of variation for metered volumes were between 0.4%-1.0% for intra-run variations and 0.5%-1.2% for inter-run variations. The aliquoting structure is compatible to common assay reagents with a wide range of liquid and material properties, demonstrated here for contact angles between 20° and 60°, densities between 789 and 1855 kg m(-3) and viscosities between 0.89 and 4.1 mPa s. The centrifugo-pneumatic multi-liquid aliquoting is implemented as a passive fluidic structure into a single fluidic layer. Fabrication is compatible to scalable fabrication technologies such as injection molding or thermoforming and does not require any additional fabrication steps such as hydrophilic or hydrophobic coatings or integration of active valves.

Centrifugal microfluidic platforms: advanced unit operations and applications.

Centrifugal microfluidics has evolved into a mature technology. Several major diagnostic companies either have products on the market or are currently evaluating centrifugal microfluidics for product development. The fields of application are widespread and include clinical chemistry, immunodiagnostics and protein analysis, cell handling, molecular diagnostics, as well as food, water, and soil analysis. Nevertheless, new fluidic functions and applications that expand the possibilities of centrifugal microfluidics are being introduced at a high pace. In this review, we first present an up-to-date comprehensive overview of centrifugal microfluidic unit operations. Then, we introduce the term "process chain" to review how these unit operations can be combined for the automation of laboratory workflows. Such aggregation of basic functionalities enables efficient fluidic design at a higher level of integration. Furthermore, we analyze how novel, ground-breaking unit operations may foster the integration of more complex applications. Among these are the storage of pneumatic energy to realize complex switching sequences or to pump liquids radially inward, as well as the complete pre-storage and release of reagents. In this context, centrifugal microfluidics provides major advantages over other microfluidic actuation principles: the pulse-free inertial liquid propulsion provided by centrifugal microfluidics allows for closed fluidic systems that are free of any interfaces to external pumps. Processed volumes are easily scalable from nanoliters to milliliters. Volume forces can be adjusted by rotation and thus, even for very small volumes, surface forces may easily be overcome in the centrifugal gravity field which enables the efficient separation of nanoliter volumes from channels, chambers or sensor matrixes as well as the removal of any disturbing bubbles. In summary, centrifugal microfluidics takes advantage of a comprehensive set of fluidic unit operations such as liquid transport, metering, mixing and valving. The available unit operations cover the entire range of automated liquid handling requirements and enable efficient miniaturization, parallelization, and integration of assays.

A microfluidic timer for timed valving and pumping in centrifugal microfluidics.

Accurate timing of microfluidic operations is essential for the automation of complex laboratory workflows, in particular for the supply of sample and reagents. Here we present a new unit operation for timed valving and pumping in centrifugal microfluidics. It is based on temporary storage of pneumatic energy and time delayed sudden release of said energy. The timer is loaded at a relatively higher spinning frequency. The countdown is started by reducing to a relatively lower release frequency, at which the timer is released after a pre-defined delay time. We demonstrate timing for 1) the sequential release of 4 liquids at times of 2.7 s ± 0.2 s, 14.0 s ± 0.5 s, 43.4 s ± 1.0 s and 133.8 s ± 2.3 s, 2) timed valving of typical assay reagents (contact angles 36-78°, viscosities 0.9-5.6 mPa s) and 3) on demand valving of liquids from 4 inlet chambers in any user defined sequence controlled by the spinning protocol. The microfluidic timer is compatible to all wetting properties and viscosities of common assay reagents and does neither require assistive equipment, nor coatings. It can be monolithically integrated into a microfluidic test carrier and is compatible to scalable fabrication technologies such as thermoforming or injection molding.

Microfluidic vapor-diffusion barrier for pressure reduction in fully closed PCR modules.

Microfluidic systems for polymerase chain reaction (PCR) should be fully closed to avoid vapor loss and to exclude the risk of contaminating the laboratory environment. In closed systems however, the high temperatures of up to 95 °C associated with PCR cause high overpressures up to 100 kPa, dominated by the increase of vapor partial pressure upon evaporation. Such high overpressures pose challenges to the mechanical stability of microfluidic chips as well as to the liquid handling in integrated sample-to-answer systems. In this work, we drastically reduce the pressure increase in fully closed PCR systems by integrating a microchannel that serves as a vapor-diffusion barrier (VDB), separating the liquid-filled PCR chamber from an auxiliary air chamber. In such configurations, propagation of vapor from the PCR chamber into the auxiliary air chamber and as a consequence the increase of pressure is limited by the slow diffusion process of vapor through the VDB. At temperature increase from 23 °C to 95 °C, we demonstrate the reduction of overpressure from more than 80 kPa without the VDB to only 35 kPa with the VDB. We further demonstrate proper function of VDB and its easy integration with downstream processes for PCR based nucleic acid amplification within centrifugal microfluidics. Without integration of the VDB, malfunction due to pressure-induced delamination of the microfluidic chip occurred.

The LabTube - a novel microfluidic platform for assay automation in laboratory centrifuges.

Assay automation is the key for successful transformation of modern biotechnology into routine workflows. Yet, it requires considerable investment in processing devices and auxiliary infrastructure, which is not cost-efficient for laboratories with low or medium sample throughput or point-of-care testing. To close this gap, we present the LabTube platform, which is based on assay specific disposable cartridges for processing in laboratory centrifuges. LabTube cartridges comprise interfaces for sample loading and downstream applications and fluidic unit operations for release of prestored reagents, mixing, and solid phase extraction. Process control is achieved by a centrifugally-actuated ballpen mechanism. To demonstrate the workflow and functionality of the LabTube platform, we show two LabTube automated sample preparation assays from laboratory routines: DNA extractions from whole blood and purification of His-tagged proteins. Equal DNA and protein yields were observed compared to manual reference runs, while LabTube automation could significantly reduce the hands-on-time to one minute per extraction.

Bubble Jet agent release cartridge for chemical single cell stimulation.

We present a new method for the distinct specific chemical stimulation of single cells and small cell clusters within their natural environment. By single-drop release of chemical agents with droplets in size of typical cell diameters (d <30 µm) on-demand micro gradients can be generated for the specific manipulation of single cells. A single channel and a double channel agent release cartridge with integrated fluidic structures and integrated agent reservoirs are shown, tested, and compared in this publication. The single channel setup features a fluidic structure fabricated by anisotropic etching of silicon. To allow for simultaneous release of different agents even though maintaining the same device size, the second type comprises a double channel fluidic structure, fabricated by photolithographic patterning of TMMF. Dispensed droplet volumes are V = 15 pl and V = 10 pl for the silicon and the TMMF based setups, respectively. Utilizing the agent release cartridges, the application in biological assays was demonstrated by hormone-stimulated premature bud formation in Physcomitrella patens and the individual staining of one single L 929 cell within a confluent grown cell culture.