Zusammenfassung Manch lebensbedrohliche Erkrankung erfordert eine schnelle Identifizierung der verantwortlichen Erreger. Solche Tests werden in spezialisierten Laboren durchgeführt, was wertvolle Zeit kostet. Deshalb entwickelt Bosch Geräte für eine Vor‐Ort‐Diagnostik zum Beispiel direkt in der Arztpraxis, die mikrofluidische Kartuschen für automatische Analysen nutzen. Die Entwicklung solcher kompakter, mobiler Geräte setzt ein tieferes Verständnis physikalischer Phänomene voraus, damit diese eine zuverlässige, einfache und schnelle Vor‐Ort‐Diagnostik ermöglichen. Das Analyseverfahren basiert auf der Polymerase‐Kettenreaktion (PCR), die eine zyklische Temperatursteuerung erfordert. Im mikrofluidischen System wird dazu die Flüssigkeit mit der Blutprobe über eine Membran zwischen geheizten und kühleren Kammer umgepumpt. Nachdem so die gesuchten DNA‐Abschnitte durch die PCR vermehrt (amplifiziert) worden sind, werden sie über Fluoreszenz optisch nachgewiesen. Dieser Artikel beschreibt eine beispielhafte Umsetzung für die molekulardiagnostische Erkennung lebensbedrohlicher Sepsis‐Infektionen anhand des Vivalytic‐Systems von Bosch.

Our microcavity array solution achieves unbiased cell isolation, rapid reagent exchange, and efficient staining for identifying tumor cells in blood.

The precision of compartment-based quantification methods is subject to multiple effects, of which partitioning and subsampling play a major role. Partitioning is the process of aliquoting the sample liquid and consequently the contained target molecules, whereas subsampling denotes the fact that usually only a portion of a sample is analyzed. In this work, we present a detailed statistical description comprising the effects of partitioning and subsampling on the relative uncertainty of the test result. We show that the state-of-the-art binomial model does not provide accurate results for the level of subsampling present when analyzing the nucleic acid content of single specific cells. Hence, in this work we address partitioning and subsampling effects separately and subsequently combine them to derive the relative uncertainty of a test system and compare it for single cell content analysis and body fluid analysis. In point-of-care test systems the area for partitioning and detection is usually limited, which means that a trade-off between the number of partitions (related to a partitioning uncertainty) and the amount of analyzed volume (related to a subsampling uncertainty) might be inevitable. In case of low target concentration, the subsampling uncertainty is dominant whereas for high target concentration, the partitioning uncertainty increases, and a larger number of partitions is beneficial to minimize the combined uncertainty. We show, that by minimizing the subsampling uncertainty in the test system, the quantification uncertainty of low target concentrations in single cell content analysis is much smaller than in body fluid analysis. In summary, the work provides the methodological basis for a profound statistical evaluation of partitioning and subsampling effects in compartment-based quantification methods and paves the way towards an improved design of future digital quantification devices for highly accurate molecular diagnostic analysis at the point-of-care.

For several decades, there has been a strong research interest in microfluidic systems and their applications. To bring these systems to market, a high development effort is often necessary for conceptualization and fabrication of such systems as well as the implementation of automated biological analysis processes. In this context, the simulation of microfluidic processes and entire microfluidic networks is becoming increasingly important, as this allows a significant reduction in development efforts, as well as easy adaptation of existing systems to specific requirements. This work presents an analytical model for elastomeric membrane-based micropumps as well as guidelines on how to apply this model to calculate microfluidic networks. The model is derived from the Young–Laplace equation for a non-prestressed elastomeric membrane with a purely nonlinear deflection as a function of applied pressure. The resulting cubic pressure–volume relation is validated by static measurements of the membrane deflection and the transported volume. The model is further used to calculate transient volume flows induced by a membrane micropump in a microfluidic network by adding Hagen Poiseuille’s law. Pressure measurements in the pump chamber confirm the basic assumptions of the model and allow definition of its validity scope. This work lays the foundation for designing elastomeric membrane-based micropumps together with microfluidic networks, estimating maximum flow rates in the system and optimizing pumping frequencies for different microfluidic configurations.

Circulating tumor cells (CTCs) that enter the bloodstream play an important role in the formation of metastases. The prognostic significance of CTCs as biomarkers obtained from liquid biopsies is intensively investigated and requires accurate methods for quantification. The purpose of this study was the capture of CTCs on an optically accessible surface for real-time quantification. A filtration device was fabricated from a transparent material so that capturing of cells could be observed microscopically. Blood samples were spiked with stained tumor cells and the sample was filtrated using a porous structure with pore sizes of 7.4 µm. The possible removal of lysed erythrocytes and the retention of CTCs were assessed. The filtration process was observed in real-time using fluorescence microscopy, whereby arriving cells were counted in order to determine the number of CTCs present in the blood. Through optimization of the microfluidic channel design, the cell retention rate could be increased by 13% (from 76% ± 7% to 89% ± 5%). Providing the possibility for real-time detection significantly improved quantification efficiency even for the smallest cells evaluated. While end-point evaluation resulted in a detection rate of 63% ± 3% of the spiked cells, real-time evaluation led to an increase of 21% to 84% ± 4%. The established protocol provides an advantageous and efficient method for integration of fully automated sample preparation and CTC quantification into a lab-on-a-chip system.

In this work, we propose a new optical measurement method and setup to investigate the dynamic behavior of a pneumatically driven diaphragm micropump in a microfluidic system. The presented method allows a contact-free spatially and temporally resolved determination of the membrane displacement. Hence, it enables to derive the volume flow rate, generated by the micropump. The method is based on the Lambert–Beer law, which describes the intensity weakening of light traveling through a medium with an absorbing substance. The fluorescence emission of a medium can thus be related to the light traveling length. The measurement method is used to deduce the flow rate profile generated by the micropump of the Lab-on-Chip system Vivalytic from Bosch Healthcare Solutions. We further quantify effects of fluidic components and system parameters on the transient flow rates. This allows the determination of maximum flow rates and pumping cycle durations as a basis for the implementation of fluidic processes on the system. The presented method requires neither additional, integrated sensor components nor a complex measurement setup. It can be implemented in any microfluidic system with membrane-based, optically accessible micropumps without major hardware modifications.

The present work deals with the microfluidic evolution of capillary surfaces that are formed during the priming of microcavity structures with a non-wetting liquid. Due to the large contact angle of the priming liquid, a trapping of air within the microcavities poses a major impediment to a complete filling. We tackle this issue by developing a two-dimensional analytical model describing the geometrical shape of capillary surfaces formed in microcavity structures. In particular, the model is employed to derive two quantitative conditions for a void-free priming of a microcavity structure in terms of its aspect ratio, rounding parameters and the channel width. Microfluidic experiments are performed to verify the analytical results. Finally, we make use of the model to demonstrate a pressure-driven aliquoting of a non-wetting sample liquid in a flow chamber with an array of 55 microcavities by introducing a second immiscible liquid acting as a sealant. In this way, our work constitutes a basis for the design of microcavity-based liquid aliquoting structures that are used in various fields of application like PCR arrays, cell culture chips or digital reaction arrays.

We report on a novel rapid prototyping approach for the manufacturing of highly individualized lab-on-chip (LoC) cartridges from generic polymer parts by laser micromachining and laser welding. The approach allows an immediate implementation of microfluidic networks, components, and functionalities into an existing LoC platform without the need for an expensive and time-consuming fabrication of production tools like molds or masks. We comprehensively describe the individual process steps of the rapid prototyping procedure including a wet-chemical treatment for an easy and effective surface polishing of laser micromachined polymer parts. For laying out, we introduce a generalized diagrammatic description of microfluidic functional units in order to design application-specific cartridges for molecular diagnostic workflows. We demonstrate the usability of our prototyped cartridges by performing microfluidic experiments within. Due to the use of generic polymer parts, our rapid prototyping approach combines a high degree of freedom with an intrinsic compatibility to an established and highly developed LoC system. By enabling an experimental testing within one day, the rapid prototyping procedure shortens development cycles and boosts the evolution of microfluidic networks as well as the implementation of novel microfluidic components and functionalities.

We report on the development of a microfluidic multiplexing technology for highly parallelized sample analysis via quantitative polymerase chain reaction (PCR) in an array of 96 nanoliter-scale microcavities made from silicon. This PCR array technology features fully automatable aliquoting microfluidics, a robust sample compartmentalization up to temperatures of 95 °C, and an application-specific prestorage of reagents within the 25 nl microcavities. The here presented hybrid silicon–polymer microfluidic chip allows both a rapid thermal cycling of the liquid compartments and a real-time fluorescence read-out for a tracking of the individual amplification reactions taking place inside the microcavities. We demonstrate that the technology provides very low reagent carryover of prestored reagents < 6 × 10 −2 and a cross talk rate < 1 × 10 −3 per PCR cycle, which facilitate a multi-targeted sample analysis via geometric multiplexing. Furthermore, we apply this PCR array technology to introduce a novel digital PCR-based DNA quantification method: by taking the assay-specific amplification characteristics like the limit of detection into account, the method allows for an absolute gene target quantification by means of a statistical analysis of the amplification results.

A novel approach to generate sequencing-ready picowell arrays requiring no emPCR and no library beads is presented. By massively-parallel digital solid-phase PCR, template DNA in length of up to 1.5 kbp is amplified and immobilized within 100,000 of 19 picoliter wells of the commercial sequencing chip (PicoTiterPlate PTPTM, Roche) in one single step. This approach has the great potential to circumvent the cumbersome state-ofthe-art emPCR workflow in the preparation of sequencing chips. Introduction Many next-generation sequencing processes [1][2] comprise four steps: (1) quantification of a DNA library, (2) DNA amplification and immobilization onto beads via emPCR, (3) sorting and arranging of beads on a surface, (4) performance of a sequencing assay. Since emPCR can be error-prone, cost-intensive, and requires multiple process steps, an alternative approach to generate sequencing-ready chips is of interest [3]. We addressed this issue by a unique approach, replacing step (2) and (3) by one single step of digital solid-phase PCR [4], directly performed in a PTP used for massively-parallel pyro-sequencing. Functional principle PCR primers are immobilized to both, the wells of an original PicoTiterPlate, and to a planar slide that seals the PTP during PCR [5]. A PCR mix containing n template DNA molecules is distributed to m wells of the sequencing chip (n < m) and sealed with the slide. The sealed sequencing chip is thermocycled generating and immobilizing PCR products only in those wells that initially contained one template molecule. For analysis of the process, immobilized PCR products are detected by sequence-specific hybridization probes. Experimental results Functionality of massively-parallel digital solid-phase PCR in PTPs is demonstrated by simultaneously amplifying DNA in length of 346 bp and 1,513 bp. Upon sequence-specific staining, the resulting two-color image shows that amplification products of both templates are successfully immobilized to the surface of the wells and the sealing slide. The distinct signals indicate the leak-tight sealing of the wells during PCR. When tenfold serial diluted template DNA is amplified by digital solid-phase PCR, the resulting number of positive wells scales with the number of initial template molecules. However, the number of positive wells is lower than expected, most probably due to adsorptive effects. When assuming a 250 × lower initial concentration, all results lie within the 95 % confidence interval of the expected Poisson distribution. This clearly indicates that positive wells result from amplification of a single molecule. Conclusion and outlook By massively-parallel digital solid-phase PCR performed in the picowell sequencing chip PTPTM, single DNA molecules are amplified and immobilized within the 19 picoliter wells and detected by hybridization. Functionality is demonstrated with template DNA as long as 1,513 bp. This is the currently smallest on-chip solid-phase PCR in a non-emulsion format. This approach has the great potential to replace emPCR in the preparation of sequencing chips saving time, costs, and material. As an outlook, our approach may enable massively-parallel single-cell analysis by isolating single cells in different wells and immobilizing specific gene products onto a substrate for further analysis. References : 1. J. Rothberg et al., Nature, 2011, 475, pp. 348. 2. M. Shirai et al., Anal. Chem., 2011, 83, pp. 7560. 3. H. Wang et al., μTAS Conf., 2011, pp. 73. 4. J. Hoffmann et al., Lab Chip, 2012, 12, pp. 3049. Cover story of volume 12 (17). 5. J. Hoffmann et al., RSC Advances, 2012, 2, pp. 3885.

A novel way of manufacturing DNA microarrays based on a copy process is presented. A picowell sequencing chip [1] is used as copy template to perform thousands of parallel PCR reactions within each 76 pl well. Thereby, the DNA from 83 % of the wells is copied onto a planar slide providing the DNA microarray copy of the sequencing chip.

We present a method for performing highly parallel PCR reactions in a picowell array (PWA) simultaneously immobilizing generated PCR products in a covalent and spatially-resolved manner onto a microscope slide via solid-phase PCR (SP-PCR). This so called PWA-SP-PCR was performed in picowell arrays featuring 100,000 wells cm(-2) of 19 pL reaction volumes with a surface-to-volume ratio of 0.2 μm(-1). Positive signals were obtained in 97.2% of the 110,000 wells in an area of 110 mm(2). Immobilized DNA was either indirectly detected using streptavidin-Cy5 or directly by molecular hybridisation of Cy3- and/or Cy5-labelled probes. Amplification and immobilization was demonstrated for template DNA ranging from 100 bp up to 1513 bp lengths. Even single DNA molecules were successfully amplified and immobilized demonstrating digital solid-phase PCR. Compared to widely established emulsion based PCR (emPCR) approaches, leading to PCR products immobilized onto bead surfaces in a highly parallel manner, the novel technique results in direct spatial registration of immobilized PCR products in a microarray format. This enables the subsequent use for massively parallel analysis similar to standard microarrays.

INTRODUCTION The perspective to automate and miniaturize assays or whole laboratories by using microfluidics or lab-on-a-chip devices led to a large research boom in microfluidics over the last two decades. However, only a limited number of commercially available devices surfaced from this boom so far. In our view, one aspect that impedes market uptake is the proprietary, complex, and expensive instrumentation that is often required to operate microfluidic chips. This approach is costly and also contributes much to the development risk.

We present a novel technique for the spatio-temporally resolved localization of liquid-gas interfaces on centrifugal microfluidic platforms based on total internal reflection (TIR) at the channel wall. The simple setup consists of a line laser and a linear image sensor array mounted in a stationary instrument. Apart from identifying the presence of usually unwanted gas bubbles, the here described online meniscus detection allows to measure liquid volumes with a high precision of 1.9%. Additionally, flow rates and viscosities (range: 1-12 mPa s, precision of 4.3%) can be sensed even during rotation at frequencies up to 30 Hz.