Advances in Microfluidics and Lab-on-a-Chip Technologies

Advances in molecular biology are enabling rapid and efficient analyses for effective intervention in domains such as biology research, infectious disease management, food safety, and biodefense. The emergence of microfluidics and nanotechnologies has enabled both new capabilities and instrument sizes practical for point-of-care. It has also introduced new functionality, enhanced sensitivity, and reduced the time and cost involved in conventional molecular diagnostic techniques. This chapter reviews the application of microfluidics for molecular diagnostics methods such as nucleic acid amplification, next-generation sequencing, high resolution melting analysis, cytogenetics, protein detection and analysis, and cell sorting. We also review microfluidic sample preparation platforms applied to molecular diagnostics and targeted to sample-in, answer-out capabilities

Wichtig ist die Work-Langeweile-Balance

Interview: 15.08.202

LabDisk employing nested PCR for rapid point-of-care diagnosis of bacterial infections

Bacterial infections still constitute one of the major healthcare burdens worldwide, despite advances in clinical diagnostics and therapy management. The conventional diagnosis of bacterial infections is mostly based on phenotypic bacterial culture, usually taking anything from half a day to several days until diagnostic results are available. These long diagnostic turnaround times are in conflict with the necessity to immediately apply antibiotic therapy as disease progression may be rapid. Consequently, antibiotic therapy is often started before laboratory results are available and definitive diagnosis can be made. This empiric antibiotic therapy initiated based on mainly unspecific clinical symptoms often leads to inappropriate use of antibiotics and is one of the major reasons for the spread of antibiotic resistances. Genotypic test methods based on the analysis of nucleic acids can significantly decrease the diagnostic turnaround time down to hours. However, these so called molecular diagnostic tests are difficult to integrate into the routine clinical laboratory workflows as they are usually complex, require substantial hands-on time and skilled personnel in a setting where human resources are often limited.The centrifugal-microfluidic “LabDisk” platform is a promising candidate for the realization of easy-to-use, automated and rapid molecular diagnostic test carriers (LabDisks) that can be operated by less trained staff, ideally “near-the-patient” in “point-of-care” analyzers. This thesis introduces a novel Polymerase-Chain-Reaction (PCR) based LabDisk, which enables rapid (~ 3-4 hours), fully automated (only 5 min hands-on time), highly sensitive (detection of During the development of the PCR-based LabDisk, technical issues of overpressure generation during PCR thermocycling and robust liquid valving were solved by introducing two novel unit operations on the LabDisk platform: Pressure reduction in fully closed microfluidic structures based on applying a vapor-diffusion-barrier (VDB) and temperature-change-rate (TCR) actuation of liquid valves. For the VDB, the overpressure in a PCR reaction chamber during a heating step of 95°C could be decreased from 80 kPa to 35 kPa, thereby preventing delamination of the sealing foil of the microfluidic structures. Robust liquid valving, a pre-requisite for the automation of molecular diagnostics on LabDisks was realized by TCR-induced pressure gradients of up to 3.5 kPa to activate a siphon valve. TCR valve actuation thereby could be implemented in a highly-integrated centrifugal-microfluidic network at no additional costs such as supplemental structures on the limited available footprint.The PCR–based LabDisk system was characterized with 200 μL spiked human serum samples, detecting bacterial pathogens Staphylococcus warneri, Streptococcus agalactiae, Escherichia coli and Haemophilus influenza down to 3 CFU, 200 CFU, 5 CFU and 2 CFU, respectively. The system was then tested with clinical serum samples from neonates with laboratory confirmed neonatal sepsis (overall 11 samples). The testing resulted in identification of 5 out of 6 bacterial species that were also detected with conventional state-of-the-art blood culture. However, compared to blood culture, multiple additional human skin-associated pathogens (mainly coagulase negative staphylococci) were identified in both the confirmed neonatal sepsis samples as well as control negative samples. These false-positive detections of bacteria were linked to traces (DNA or bacterical cells) of human-skin associated bacteria in the LabDisk, resulting from the non-DNA-free fabrication of the LabDisks. In further experiments, LabDisk tests with urine samples from patients with laboratory-confirmed urinary tract infection (overall 4 samples) and a control negative sample showed a perfect match to the results of urine culture.Finally, a complementary immunoassay LabDisk was developed that can also be processed with the LabDisk player. It enables analysis of both, DNA and protein biomarkers on a single processing device. Human C-reactive protein (CRP), an important inflammatory marker in bacterial infections, can be quantified in human serum in the pathophysiological range i.e. 3 – 81 ng/mL with a limit of detection and limit of quantification of 1.5 ng/mL and 1.8 ng/mL, respectively. The test features an analysis time of ~ 25 min and is processed in a fully automated manner after loading of the sample and immunoreagents, requiring approximately 5 min hands-on time.With further advancements of the two carriers, including prestorage of all required liquid reagents, molecular diagnostics and protein biomarker analysis could be realized at the point-of-care. This could further reduce analytical turnaround times without the necessity to transport samples to the laboratory. The tests could be integrated more easily into the routine clinical workflow and would enable targeted evidence-based antibiotic therapy at much earlier stages. This may lead to better patient outcomes for bacterial infections and less (mis)use of ineffective antibiotic agents, therewith fighting the spread of antimicrobial resistances

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

A centrifugal microfluidic method with pre-stored reagents for automation of particle based immunoassays controlled by the spinning frequency only.</p