Development of a Fluorescent Microfluidic Device Based on Antibody Microarray Read-Out for Therapeutic Drug Monitoring of Acenocoumarol.

The development of a proof-of-concept point-of-care (PoC) device for the determination of oral anticoagulants determination is presented. Acenocoumarol (ACL) is prescribed to prevent certain cardiovascular diseases related to the prevention of deep vein thrombosis, pulmonary embolism, myocardial infarction, and stroke. Oral anticoagualant treatment (OAT) represents a population of 2% under treatment which has expenditures about $ 144 million in 2011. The main drawback for OAT is the associated narrow therapeutic window and the unpredictable dose-response relationship, which is one of the main causes for visiting the emergency room at the hospitals. In a previous work, family antibodies were produced for the simultaneous detection of ACL and warfarin (W) depending on the area of application. It was developed in different formats, indirect and direct, either with similar detectabilities and both assays quantifying the oral anticoagulants with high accuracy and reproducibility. We present the implementation of the already developed immunochemical method to a point-of-care (PoC) device to assist on the patient compliance assessment programs. In order to achieve this goal, a first development was performed implementing ACL ELISA assay into a microarray format with fluorescent read-out. The assay was successfully implemented achieving a LOD of 1.23 nM of ACL directly measured in human plasma. Then, a fully integrated microfluidic system is developed which incorporates the specific immunoreagents for the detection of ACL. The immunoreagents were attached onto the glass slide in a microarray format. The system is automatic, rapid, sensitive, and disposable that could help clinicians monitor patients under OAT. According to the fluorescent label of the ACL binding, the chip can be easily read with a scanner. The microfluidic system performed good according to the robust and reproducible signals, and subsequently yielded an accurate result.

Liquids on-chip: direct storage and release employing micro-perforated vapor barrier films.

Liquids on-chip describes a reagent storage concept for disposable pressure driven Lab-on-Chip (LoC) devices, which enables liquid storage in reservoirs without additional packaging. On-chip storage of liquids can be considered as one of the major challenges for the commercial break through of polymer-based LoC devices. Especially the ability for long-term storage and reagent release on demand are the most important aspects for a fully developed technology. On-chip storage not only replaces manual pipetting, it creates numerous advantages: fully automated processing, ease of use, reduction of contamination and transportation risks. Previous concepts for on-chip storage are based on liquid packaging solutions (e.g. stick packs, blisters, glass ampoules), which implicate manufacturing complexity and additional pick and place processes. That is why we prefer on-chip storage of liquids directly in reservoirs. The liquids are collected in reservoirs, which are made of high barrier polymers or coated by selected barrier layers. Therefore, commonly used polymers for LoC applications as cyclic olefin polymer (COP) and polycarbonate (PC) were investigated in the context of novel polymer composites. To ensure long-term stability the reservoirs are sealed with a commercially available barrier film by hot embossing. The barrier film is structured by pulsed laser ablation, which installs rated break points without affecting the barrier properties. A flexible membrane is actuated through pneumatic pressure for reagent release on demand. The membrane deflection breaks the barrier film and leads to efficient cleaning of the reservoirs in order to provide the liquids for further processing.

Force measurement in the presence of Brownian noise: equilibrium-distribution method versus drift method.

The study of microsystems and the development of nanotechnologies require alternative techniques to measure piconewton and femtonewton forces at microscopic and nanoscopic scales. Among the challenges is the need to deal with the ineluctable thermal noise, which, in the typical experimental situation of a spatial diffusion gradient, causes a spurious drift. This leads to a correction term when forces are estimated from drift measurements [G. Volpe, L. Helden, T. Brettschneider, J. Wehr, and C. Bechinger, Phys. Rev. Lett. 104, 170602 (2010)]. Here we provide a systematic study of such an effect by comparing the forces acting on various Brownian particles derived from equilibrium-distribution and drift measurements. We discuss the physical origin of the correction term, its dependence on wall distance and particle radius, and its relation to the convention used to solve the respective stochastic integrals. Such a correction term becomes more significant for smaller particles and is predicted to be on the order of several piconewtons for particles the size of a biomolecule.

Influence of noise on force measurements.

We demonstrate how the ineluctable presence of thermal noise alters the measurement of forces acting on microscopic and nanoscopic objects. We quantify this effect exemplarily for a Brownian particle near a wall subjected to gravitational and electrostatic forces. Our results demonstrate that the force-measurement process is prone to artifacts if the noise is not correctly taken into account.

Novel perspectives for the application of total internal reflection microscopy.

Total Internal Reflection Microscopy (TIRM) is a sensitive non-invasive technique to measure the interaction potentials between a colloidal particle and a wall with femtonewton resolution. The equilibrium distribution of the particle-wall separation distance z is sampled monitoring the intensity I scattered by the Brownian particle under evanescent illumination. Central to the data analysis is the knowledge of the relation between I and the corresponding z, which typically must be known a priori. This poses considerable constraints to the experimental conditions where TIRM can be applied (short penetration depth of the evanescent wave, transparent surfaces). Here, we introduce a method to experimentally determine I(z) by relying only on the distance-dependent particle-wall hydrodynamic interactions. We demonstrate that this method largely extends the range of conditions accessible with TIRM, and even allows measurements on highly reflecting gold surfaces where multiple reflections lead to a complex (z).