Lab-on-a-disk extraction of PBMC and metered plasma from whole blood: An advanced event-triggered valving strategy.

In this paper, we present a centrifugal microfluidic concept employing event-triggered valving for automated extraction of metered plasma and peripheral blood mononuclear cells (PBMCs). This "lab-on-a-disk" system has been developed for retrieving different density layers from a liquid column by "overflowing" the layers sequentially using the pressure exerted by a density-gradient liquid. Defined volumes of plasma and PBMCs were efficiently forwarded into designated microfluidic chambers as a sample preparation step prior to further downstream processing. Furthermore, the extracted PBMCs were counted directly on-disk using an automated optical unit by object-based image analysis, thus eliminating the requirement for the post-processing of the extracted PBMCs. This study is a direct continuation of our previous work1 where we demonstrated combined on-disk detection of C-reactive protein and quantification of PBMCs following on-disk extraction of plasma and PBMCs from a single blood sample using a centrifugo-pneumatic valving mechanism. However, the former valving technique featured limited PBMC extraction efficiency. Here, integrating the novel concept along with event-triggered valving mechanism, we eliminated the occurrence of a specific microfluidic effect, which led us to increase PBMC extraction efficiency to 88%. This extraction method has the potential to be utilized for efficiently separating multiple density layers from a liquid sample in relevant biomedical applications.

New Evidence for the Mechanism of Action of a Type-2 Diabetes Drug Using a Magnetic Bead-Based Automated Biosensing Platform.

The mechanism of action (MOA) of the first line type-2 diabetes drug metformin remains unclear despite its widespread usage. However, recent evidence suggests that the mitochondrial copper (Cu)-binding action of metformin may contribute toward the drug's MOA. Here, we present a novel biosensing platform for investigating the MOA of metformin using a magnetic microbead-based agglutination assay which has allowed us to demonstrate for the first time the interaction between Cu and metformin at clinically relevant low micromolar concentrations of the drug, thus suggesting a potential pathway of metformin's blood-glucose lowering action. In this assay, cysteine-functionalized magnetic beadswere agglutinated in the presence of Cu due to cysteine's Cu-chelation property. Addition of clinically relevant doses of metformin resulted in disaggregation of Cu-bridged bead-clusters, whereas the effect of adding a closely related but blood-glucose neutral drug propanediimidamide (PDI) showed completely different responses to the clusters. The entire assay was integrated in an automated microfluidics platform with an advanced optical imaging unit by which we investigated these aggregation-disaggregation phenomena in a reliable, automated, and user-friendly fashion with total assay time of 17 min requiring a sample (metformin/PDI) volume of 30 µL. The marked difference of Cu-binding action between the blood-glucose lowering drug metformin and its inactive analogue PDI thus suggests that metformin's distinctive Cu-binding properties may be required for its effect on glucose homeostasis. The novel automated platform demonstrating this novel investigation thus holds the potential to be utilized for investigating significant and sensitive molecular interactions via magnetic bead-based agglutination assay.

Lab-on-a-disc agglutination assay for protein detection by optomagnetic readout and optical imaging using nano- and micro-sized magnetic beads.

We present a biosensing platform for the detection of proteins based on agglutination of aptamer coated magnetic nano- or microbeads. The assay, from sample to answer, is integrated on an automated, low-cost microfluidic disc platform. This ensures fast and reliable results due to a minimum of manual steps involved. The detection of the target protein was achieved in two ways: (1) optomagnetic readout using magnetic nanobeads (MNBs); (2) optical imaging using magnetic microbeads (MMBs). The optomagnetic readout of agglutination is based on optical measurement of the dynamics of MNB aggregates whereas the imaging method is based on direct visualization and quantification of the average size of MMB aggregates. By enhancing magnetic particle agglutination via application of strong magnetic field pulses, we obtained identical limits of detection of 25pM with the same sample-to-answer time (15min 30s) using the two differently sized beads for the two detection methods. In both cases a sample volume of only 10µl is required. The demonstrated automation, low sample-to-answer time and portability of both detection instruments as well as integration of the assay on a low-cost disc are important steps for the implementation of these as portable tools in an out-of-lab setting.

The copper binding properties of metformin--QCM-D, XPS and nanobead agglomeration.

Study of the copper binding properties of metformin is important for revealing its mechanism of action as a first-line type-2 diabetes drug. A quantitative investigation of interactions between metformin and L-cysteine-copper complexes was performed. The results suggest that metformin could interact with biological copper, which plays a key role in mitochondrial function.