The development of new drugs is time-consuming, extremely expensive and often promising drug candidates fail in late stages of the development process due to the lack of suitable tools to either predict toxicological effects or to test drug candidates in physiologically relevant environments prior to clinical tests. We therefore try to develop diagnostic multiorgan microfluidic chips based on patient specific induced pluripotent stem cell (iPS) technology to explore liver dependent toxic effects of drugs on individual human tissues such as liver or kidney cells. Based initially on standardized microfluidic modules for cell culture, we have developed integrated microfluidic devices which contain different chambers for cell/tissue cultivation. The devices are manufactured using injection molding of thermoplastic polymers such as polystyrene or cyclo-olefin polymer. In the project, suitable surface modification methods of the used materials had to be explored. We have been able to successfully demonstrate the seeding, cultivation and further differentiation of modified iPS, as shown by the use of differentiation markers, thus providing a suitable platform for toxicity testing and potential tissue-tissue interactions.
A central method in a standard biochemical laboratory is represented by the polymerase chain reaction (PCR), therefore
many attempts have been performed so far to implement this technique in lab-on-a-chip (LOC) devices. PCR is an ideal
candidate for miniaturization because of a reduction of assay time and decreased costs for expensive bio-chemicals. In
case of the “classical” PCR, detection is done by identification of DNA fragments electrophoretically separated in
agarose gels. This method is meanwhile frequently replaced by the so-called Real-Time-PCR because here the
exponential increase of amplificates can be observed directly by measurement of DNA interacting fluorescent dyes. Two
main methods for on-chip PCRs are available: traditional “batch” PCR in chambers on a chip using thermal cycling,
requiring about 30 minutes for a typical PCR protocol and continuous-flow PCR, where the liquid is guided over
stationary temperature zones. In the latter case, the PCR protocol can be as fast as 5 minutes. In the presented work, a
proof of concept is demonstrated for a real-time-detection of PCR products in microfluidic systems.
There is a need to design an integrated microfluidic platform as simple and lean as possible in order to meet the
requirements for a miniaturized system. Magnetic particles show a great versatility in performing several of the functions
necessary in many microfluidic assays. We therefore have developed a compact portable system to perform magneticbead-
based sample preparation steps in a chip such as DNA-extraction or particle-enhanced mixing of reagents. A
central application in a standard biochemical/biological/medical laboratory is represented by PCR. The execution of a
cyclic heating profile during PCR is a considerable stress for chip and liquid inside the chip because evaporation and
uncontrolled condensation or unintended motion of the PCR solution.
One strategy to overcome this problem consists of the implementation of valves flanking a stationary PCR in appropriate
incubation cavities. In addition to the well-known elastomeric membrane valves, wax-valves mechanical turning or
rotary valves flanking the PCR chamber, we present in this paper the use of clustered magnetic particles as blocking
valves for such reaction chambers.
We report on the capability of assembled magnetic particles to act as rather simple configurated valves during a PCR
typical temperature regime. These novel valves efficiently withstand 1.5 bar pressure, prevent loss of aqueous liquid
inside the reaction chamber via evaporation or bubble formation, and do not express adverse effects on any biological
reaction inside the chip-based PCR cavity. The latter properties have been proven by a set of different PCRs performed
in chip-based cavities.
Taking advantage of microfluidics technology, a Lab-on-Chip system was developed offering the possibility of
performing HLA (Human Leukocyte Antigen) typing to test genetic predisposition to coeliac disease and measure the
level of immunodeficiency at the point-of-care. These analysis procedures are implemented on two different microfluidic
cartridges, both having identical interfacial connections to the identical automated instrument.
In order to assess the concentration of the targeted analytes in human blood, finger prick samples are processed to either
extract genomic DNA carrying the coeliac disease gene or blood plasma containing the disease specific antibodies. We
present here the different microfluidic modules integrated in a common platform, capable of automated sample
preparation and analyte detection.
In summary, this new microfluidic approach will dramatically reduce the costs of materials (polymer for the disposable
chips and minute amount of bio-reagents) and minimize the time for analysis down to less than 20 minutes. In
comparison to the state of the art detection of coeliac disease this work represents a tremendous improvement for the
patient's quality of live and will significantly reduce the cost burden on the health care system.
Alex Fragoso, Noemi Laboria, Mary Luz Botero, Diego Bejarano, Daniel Latta, Thomas Hansen-Hagge, Wolfgang Kemmner, Ioanis Katakis, Claudia Gärtner, Klaus Drese, Ciara O'Sullivan
A microsystem integrating electrochemical biosensoric detection for the simultaneous multiplexed detection of protein
markers of breast cancer is reported. The immobilization of antibodies against each of carcinoembryonic antigen (CEA),
prostate specific antigen (PSA) and cancer antigen 15-3 (CA15-3) was achieved via crosslinking to a bipodal dithiol
chemisorbed on gold electrodes. This bipodal dithiol had the double function of eliminating non-specific binding and
optimal spacing of the anchor antibodies for maximum accessibility to the target proteins. Storage conditions were
optimized, demonstrating a long-term stability of the reporter conjugates jointly stored within a single reservoir in the
microsystem. The final system has been optimized in terms of incubation times, temperatures and simultaneous,
multiplexed detection of the protein markers was achieved in less than 10 minutes with less than ng/mL detection limits.
The microsystem has been validated using real patient serum samples and excellent correlation with ELISA results
obtained.
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