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.

Integrated microfluidic platform for the electrochemical detection of breast cancer markers in patient serum samples.

A microsystem integrating electrochemical detection for the simultaneous detection of protein markers of breast cancer is reported. The microfluidic platform was realized by high precision milling of polycarbonate sheets and features two well distinguishable sections: a detection zone incorporating the electrode arrays and the fluid storage part. The detection area is divided into separate microfluidic chambers addressing selected electrodes for the measurement of samples and calibrators. The fluidic storage part of the platform consists of five reservoirs to store the reagents and sample, which are interfaced by septa. These reservoirs have the appropriate volume to run a single assay per cartridge and are manually filled. The liquids from the reservoirs are actuated by applying a positive air pressure (i.e.via a programmable syringe pump) through the septa and are driven to the detection zone via two turning valves. The application of the realised platform in the individual and simultaneous electrochemical detection of proteic cancer markers with very low detection limits are demonstrated. The microsystem has also been validated using real patient serum samples and excellent correlation with ELISA results obtained.

Compact, cost-efficient microfluidics-based stopped-flow device.

Stopped-flow technology is frequently used to monitor rapid (bio)chemical reactions with high temporal resolution, e.g., in dynamic investigations of enzyme reactions, protein interactions, or molecular transport mechanisms. However, conventional stopped-flow devices are often overly complex, voluminous, or costly. Moreover, excessive amounts of sample are often wasted owing to inefficient designs. To address these shortcomings, we propose a stopped-flow system based on microfluidic design principles. Our simple and cost-efficient approach offers distinct advantages over existing technology. In particular, the use of injection-molded disposable microfluidic chips minimizes required sample volumes and associated costs, simplifies handling, and prevents adverse cross-contamination effects. The cost of the system developed is reduced by an order of magnitude compared with the cost of commercial systems. The system contains a high-precision valve system for fluid control and features automated data acquisition capability with high temporal resolution. Analyses with two well-established reaction kinetics yielded a dead time of approximately 8-9 ms.

Transmission of oxLDL-derived lipid peroxide radicals into membranes of vascular cells is the main inducer of oxLDL-mediated oxidative stress.

Oxidatively modified LDL is generally accepted to be an important elicitor of pro-mitotic, pro-inflammatory, and atherogenic effects in vascular cells. The uptake of oxLDL and concomitant activation of the O(2)*-producing NAD(P)H oxidase and/or oxLDL as a self-contained emitter of O(2)* are believed to trigger these malfunctions. The following observations allowed reinvestigating the mode of oxLDL-induced stress: (1) we observed that artery smooth muscle primary cells internalize fluorescently labelled oxidized or acetylated LDL considerably less efficient than endothelial cells. (2) Both types of cells, however, displayed an oxLDL concentration dependent level of oxidative stress as monitored by the oxidation of carboxy-H2DCFDA to fluorescent carboxy-DCF. A dose dependent decrease of dihydroethidine oxidation to oxyethidine implied an oxLDL-induced depletion of the cellular energy pool. The release of O(2)* by exogenous oxLDL, as postulated above, did not sufficiently explain intracellular stress because the fluorescence was only marginally blocked by antioxidative enzymes (SOD, catalase) or substances (L-NAME, DMSO, DMHP, DMTU). We were able to reveal a third mode of oxLDL-induced stress by showing with the help of a fluorescent, oxidizable lipid analogue (BODIPY 581/591 C(11)) that oxLDL-derived lipid peroxides and radicals migrate into cellular membranes giving rise to a chronic inoculation of the vascular cells with oxidative chain reactions. The novel data may help to design adequate therapeutic strategies against oxLDL-induced cardiovascular diseases.