We present a novel, cost-efficient process chain for fast tooling and small-lot replication of high-quality, multi-scale microfluidic polymer chips within less than 5 days. The fabrication chain starts with a primary master which is made by well-established cleanroom processes such as DRIE or negative SU-8 resist based surface micromachining. The formation of undercuts in the master which would complicate demolding is carefully avoided. Secondary PDMS masters or epoxy-based masters which are more suitable for common polymer replication schemes such as soft-embossing, hot-embossing or injection molding are subsequently cast from the primary masters. The polymer replica are mainly made of COC and show excellent fidelity with the conventionally micromachined master while displaying no degeneration, even after more than 200 cycles. The use of other polymers such as PMMA is also possible. The process chain further includes surface modification techniques for overall, long-term stable hydrophilic coatings and for local hydrophobic patches as well as a durable sealing based on thermal bonding.
In contrast to DNA microarrays, production of protein microarrays is an immense technological challenge due to high complexity and diversity of proteins. In this paper we investigate three essential aspects of protein microarray fabrication based on the highly parallel and non-contact TopSpot technology: evaporation of probes during long lasting production times, optimization of protein immobilization and improvement of protein microarray reproducibility. Evaporation out of the printhead reservoirs was reduced to a minimum by sealing the reservoirs with gas permeable foils or PDMS frames. This led to dramatically lowered setup times through the possibility of long-term, ready-to-print storage of filled printheads. To optimize immobilization efficiency 128 printing buffers were tested by printing two different proteins onto seven different microarray slide types. This way we were able to reduce the CV of spot diameter on the microarray slide below 1.14%. To remarkably increase protein immobilization efficiency on microarray slides the commonly used EDC-NHS system (a laboratory method for immobilization of proteins) was miniaturized by using a new drop-in-drop printing technique. Additionally the very fast UV cross-linking was used to immobilize antibodies. The optimized system was used to produce antibody microarrays and with it microarray ELISA experiments were performed successfully.
We present a novel concept to process human blood on a spinning polymer disk for the determination of the hematocrit level by simple visual inspection. The microfluidic disk which is spun by a macroscopic drive unit features an upstream metering structure and a downstream blind channel where the centrifugally enforced sedimentation of the blood is performed. The bubble-free priming of the blind channel is governed by centrifugally assisted capillary filling along the sloped hydrophilic side-wall and the lid as well as the special shape of the dead end of the two-layer channel. The hematocrit is indicated at the sharp phase boundary between the plasma and the segregated cellular pellet on a disk-imprinted calibrated scale. This way, we conduct the hematocrit determination of human blood within 5 min at a high degree of linearity (R(2) = 0.999) and at a high accuracy (CV = 4.7%) spanning over the physiological to pathological working range. As all processing steps including the priming, the metering to a defined volume as well as the centrifugation are executed automatically during rotation, the concept is successfully demonstrated in a conventional PC-CDROM drive while delivering the same performance (R(2) = 0.999, CV = 4.3%).
We report about the correlation between satellite free droplet release and liquid viscosity in a highly parallel, pressure driven nanoliter dispenser. In extensive studies, we found that for liquids of different viscosities the duration of the pressure pulse is the predominant effect compared to pressure amplitude. This result is of essential importance when actuation parameters have to be adopted for different media like oligonucleotide, DNA or protein solutions as it is the case for the non-contact high throughput fabrication of microarrays (Ducree et al., 2000). Experiments with oligonucleotides as well as with different proteins showed ascertained carry-over and cross-contamination free printing of DNA and protein microarrays. With it a prime critical point of microarray production is solved, leading to high quality whilst high throughput microarray fabrication. For oligonucleotides printing, we found CVs to be better than 1% within one single dispensing channel and 1.5% within all 24 channels of a 24 channel printhead for each used printing buffer. By optimizing the protein printing buffer the CVs for protein printing were reduced to about 1% within all 24 channels. As a serious practical application test oligonucleotides microarrays were produced using our nanoliter dispenser system. With it a full DNA hybridization experiment was performed. Clear positive signals one hand and no signals in the negative controls on the other hand showed that our system is suited for microarray production.
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