Marion Ritzi-Lehnert

Development of chip-compatible sample preparation for diagnosis of infectious diseases

Diagnosis of infectious diseases in primary care is predominantly based on medical history and physical examination, as conventional laboratory investigations are often associated with delays that are unacceptable in medical practice. Point-of-care testing, and especially lab-on-a-chip (LoC) systems, are expected to result in a considerable reduction in associated healthcare costs and lead to fast, but appropriate and effective, personalized therapy. Although appropriate sample preparation is essential for final detection, most microfluidic-based approaches start from samples prepared by conventional laboratory procedures, therefore continuing to restrict the use of these systems to a laboratory setting. The lack of integrated sample preparation, especially for sample volumes in the milliliter range, is a major drawback of existing LoC systems. LoC systems that start with real samples and perform a full protocol from sample to result are still rare. In this article, the most recent advances in on-chip sample preparation are reviewed for microfluidic-based diagnosis of infectious diseases.

Fast nucleic acid amplification for integration in point‐of‐care applications

An ultrafast microfluidic PCR module (30 PCR cycles in 6 min) based on the oscillating fluid plug concept was developed. A robust amplification of native genomic DNA from whole blood samples could be achieved at operational conditions established from systematic investigations of key parameters including heat transfer and in particular flow velocities. Experimental data were augmented with results from computational fluid dynamics simulations. The reproducibility of the current system was substantially improved compared to previous concepts by integration of a closed reservoir instead of utilizing a vented channel end at ambient pressure rendering the devised module suitable for integration into complex sample‐to‐answer analysis platforms such as point‐of‐care applications.

Automated genotyping of circulating tumor cells

Cancer remains a prominent health concern in modern societies. Continuous innovations and introduction of new technologies are essential to level or reduce current healthcare spending. A diagnostic platform to detect circulating tumor cells (CTCs) in peripheral blood may be most promising in this respect. CTCs have been proposed as a minimally invasive, prognostic and predictive marker to reflect the biological characteristics of tumors and are implemented in an increasing number of clinical studies. Still, their detection remains a challenge as they may occur at concentrations below one single cell per ml of blood. To facilitate their detection, here we describe microfluidic modules to isolate and genotype CTCs directly from clinical blood samples. In a first cell isolation and detection module, the CTCs are immunomagnetically enriched, separated and counted. In a second module and after cell lysis, the mRNA is reversely transcripted to cDNA, followed by a multiplex ligation probe amplification of 20 specific genetic markers and two control fragments. Following the multiplex ligation probe amplification reaction, the amplified fragments are electrochemically detected in a third and final module. Besides the design of the modules, their functionality is described using control samples. Further testing using clinical samples and integration of all modules in a single, fully automated smart miniaturized system will enable minimal invasive testing for frequent detection and characterization of CTCs.

Automated DNA-preparation system for bacteria out of air sampler liquids

Preventing bacterial contaminations is a significant challenge in applications across a variety of industries, e.g. in food processing, the life sciences or biohazard detection. Here we present a fully automated lab-on-a-chip system wherein a disposable microfluidic chip moulded by polymeric injection is inserted into an operating device. Liquid samples, here obtained from an air sampler, can be processed to extract and lyse bacteria, and subsequently to purify their DNA using a silica matrix. After the washing and elution steps, the DNA solution is dispensed into a reaction vessel for further analysis in a conventional laboratory polymerase chain reaction (PCR) device. We demonstrate the workability and efficiency of our approach with results from a 9 ml liquid sample spiked with E. coli.

Justification of rapid prototyping in the development cycle of thermoplastic-based lab-on-a-chip†

During the developmental cycle of lab‐on‐a‐chip devices, various microstructuring techniques are required. While in the designing and assay implementation phase direct structuring or so‐called rapid‐prototyping methods such as milling or laser ablation are applied, replication methods like hot embossing or injection moulding are favourable for large quantity manufacturing. This work investigated the applicability of rapid‐prototyping techniques for thermoplastic chip development in general, and the reproducibility of performances in dependency of the structuring technique. A previously published chip for prenatal diagnosis that preconcentrates DNA via electrokinetic trapping and field‐amplified‐sample‐stacking and afterwards separates it in CGE was chosen as a model. The impact of structuring, sealing, and the integration of membranes on the mobility of the EOF, DNA preconcentration, and DNA separation was studied. Structuring methods were found to significantly change the location where preconcentration of DNA occurs. However, effects on the mobility of the EOF and the separation quality of DNA were not observed. Exchange of the membrane has no effect on the chip performance, whereas the sealing method impairs the separation of DNA within the chip. The overall assay performance is not significantly influenced by different structuring methods; thus, the application of rapid‐prototyping methods during a chip development cycle is well justified.

Numerical Study of Micromixers for Stopped Flow

Computational fluid dynamics (CFD) simulations are conducted to compare the efficiency of different types of micromixers with respect to their potential application in chip-based stopped-flow designs. The evaluation of a mixing parameter for different geometries and at various flow velocities enables a comparison between the Berger ball mixer and other designs that are in principle suitable for applications in microfluidic chips.Copyright

New Lab-on-a-Chip System for Infectious Disease Analysis

Early diagnosis followed by personalised efficient therapy of infectious diseases (e.g. respiratory diseases, meningitis, sepsis) can lead to considerable reduction of costs in health care. Point-of-care testing (POCT) can provide early detection since this kind of decentralised analysis can be done by unskilled personnel at any time. Other advantages of automated miniaturised Lab-on-a-Chip systems (LoC) are reduction of time and reagents, elimination of cross-contamination and enhanced reproducibility due to enhanced process control. Such Lab-on-a-Chip systems will establish themselves on market only when sensitivity and specificity meet clinical requirements. An integrated cost-efficient lab-on-a-chip system is presented which allows performing all diagnostic process steps for pathogen analysis of respiratory viruses from nasopharyngeal samples. The microfluidic disposable chip comprises structures for lysis of nasopharyngeal swab samples, preparation of total nucleic acids using magnetic silica beads, reverse transcription followed by QIAplex PCR technology and labelling of the nucleic acids by hybridisation with LiquiChip Beads and streptavidin-R-phycoerythrin. Labelled target sequences are transferred for analysis into a QIAGEN LiquiChip 200 workstation. The core of the instrument is a construction based on rotating heating bars allowing for fast cycling. All chemicals needed for performing of 24 analyses are either stored freeze-dried on the single-use disposable microfluidic chip (processing cartridge) or as liquids in a separate reagent cartridge. After introducing the sample into the lysis chamber of the microfluidic chip and inserting the chip into the device all steps are done automatically. To realise these steps, fluidic control in terms of light barriers and turning valves are integrated into the injection moulded disposable chip. This includes metering structures as well as magnetic stir bars for mixing. The functionality was proven by direct comparison of samples processed manually vs. automatically using the “ResPlex Panel II” for detection of respiratory viruses from nasopharyngeal samples. The efficiency of the automated LoC system yields at about 30–60% as compared to the manually performed reference experiments. Comparing the performance of the instrument with commercially available kits and nucleic acid preparation devices showed slightly weaker but clearly positive final signal intensities obtained from the prototype device even without protocol optimization.Copyright