Microfluidic Stress Device to Decouple the Synergistic Effect of Shear and Interfaces on Antibody Aggregation.

Protein denaturation and aggregation resulting from the effects of interfacial stress, often enhanced by flow and shear stress, pose significant challenges in the production of therapeutic proteins and monoclonal antibodies. The influence of flow on protein stability is closely intertwined with interfacial effects. In this study, we have developed a microfluidic device capable of exposing low volume (< 320 µL) protein solutions to highly uniform shear. To disentangle the synergistic impact of flow and interfaces on protein aggregation, we fabricated two devices composed of different materials, namely poly(methyl methacrylate) (PMMA) and stainless steel. Upon application of shear, we observed formation of protein particles in the micron-size range. Notably, The number of particles generated in the steel devices was ∼ 3.5 fold lower than in the PMMA device, hinting at an interface-mediated effect. With increasing the protein concentration from 1 to 50 mg/mL we observed a saturation in the amount of aggregates, further confirming the key role of solid-liquid interfaces in inducing particle formation. Introduction of non-ionic surfactants prevented protein aggregation, even at the highest tested protein concentration and low surfactant concentrations of 0.05 mg/mL. Overall, our findings corroborate the synergistic impact of shear and interface effects on protein aggregation. The device developed in this study offers a small-scale platform for assessing the stability of antibody formulations throughout various stages of the development and manufacturing process.

High Aspect Ratio Nanoscale Pores through BCP-Based Metal Oxide Masks and Advanced Dry Etching.

The reliable and regular modification of the surface properties of substrates plays a crucial role in material research and the development of functional surfaces. A key aspect of this is the development of the surface pores and topographies. These can confer specific advantages such as high surface area as well as specific functions such as hydrophobic properties. Here, we introduce a combination of nanoscale self-assembled block-copolymer-based metal oxide masks with optimized deep reactive ion etching (DRIE) of silicon to permit the fabrication of porous topographies with aspect ratios of up to 50. Following the evaluation of our procedure and involved parameters using various techniques, such as AFM or SEM, the suitability of our features for applications relying on high light absorption as well as efficient thermal management is explored and discussed in further detail.

Optical feedback control loop for the precise and robust acoustic focusing of cells, micro- and nanoparticles.

Despite a long history and the vast number of applications demonstrated, very few market products incorporate acoustophoresis. Because a human operator must run and control a device during an experiment, most devices are limited to proof of concepts. On top of a possible detuning due to temperature changes, the human operator introduces a bias which reduces the reproducibility, performance and reliability of devices. To mitigate some of these problems, we propose an optical feedback control loop that optimizes the excitation frequency. We investigate the improvements that can be expected when a human operator is replaced for acoustic micro- and nanometer particle focusing experiments. Three experiments previously conducted in our group were taken as a benchmark. In addition to being automatic, this resulted in the feedback control loop displaying a superior performance compared to an experienced scientist in 1) improving the particle focusing by at least a factor of two for 5 µm diameter PS particles, 2) increasing the range of flow rates in which 1 µm diameter PS particles could be focused and 3) was even capable of focusing 600 nm diameter PS particles at a frequency of 1.72075 MHz. Furthermore, the feedback control loop is capable of focusing biological cells in one and two pressure nodes. The requirements for the feedback control loop are: an optical setup, a run-of-the-mill computer and a computer controllable function generator. Thus resulting in a cost-effective, high-throughput and automated method to rapidly increase the efficiency of established systems. The code for the feedback control loop is openly accessible and the authors explicitly wish that the community uses and modifies the feedback control loop to their own needs.

Embedded Microbubbles for Acoustic Manipulation of Single Cells and Microfluidic Applications.

Acoustically excited microstructures have demonstrated significant potential for small-scale biomedical applications by overcoming major microfluidic limitations. Recently, the application of oscillating microbubbles has demonstrated their superiority over acoustically excited solid structures due to their enhanced acoustic streaming at low input power. However, their limited temporal stability hinders their direct applicability for industrial or clinical purposes. Here, we introduce the embedded microbubble, a novel acoustofluidic design based on the combination of solid structures (poly(dimethylsiloxane)) and microbubbles (air-filled cavity) to combine the benefits of both approaches while minimizing their drawbacks. We investigate the influence of various design parameters and geometrical features through numerical simulations and experimentally evaluate their manipulation capabilities. Finally, we demonstrate the capabilities of our design for microfluidic applications by investigating its mixing performance as well as through the controlled rotational manipulation of individual HeLa cells.

Reduced Etch Lag and High Aspect Ratios by Deep Reactive Ion Etching (DRIE).

Deep reactive ion etching (DRIE) with the Bosch process is one of the key procedures used to manufacture micron-sized structures for MEMS and microfluidic applications in silicon and, hence, of increasing importance for miniaturisation in biomedical research. While guaranteeing high aspect ratio structures and providing high design flexibility, the etching procedure suffers from reactive ion etching lag and often relies on complex oxide masks to enable deep etching. The reactive ion etching lag, leading to reduced etch depths for features exceeding an aspect ratio of 1:1, typically causes a height difference of above 10% for structures with aspect ratios ranging from 2.5:1 to 10:1, and, therefore, can significantly influence subsequent device functionality. In this work, we introduce an optimised two-step Bosch process that reduces the etch lag to below 1.5%. Furthermore, we demonstrate an improved three-step Bosch process, allowing the fabrication of structures with 6 µm width at depths up to 180 µm while maintaining their stability.

Manipulation of single cells inside nanoliter water droplets using acoustic forces.

Droplet microfluidics enables high-throughput screening of single cells and is particularly valuable for applications, where the secreted compounds are analyzed. Typically, optical methods are employed for analysis, which are limited in their applicability as labeling protocols are required. Alternative label-free methods such as mass spectrometry would broaden the range of assays but are harmful to the cells, which is detrimental for some applications such as directed evolution. In this context, separation of cells from supernatant is beneficial prior to the analysis to retain viable cells. In this work, we propose an in-droplet separation method based on contactless and label-free acoustic particle manipulation. In a microfluidic chip, nanoliter droplets containing particles are produced at a T-junction. The particles are trapped in the tip of the droplet by the interplay of acoustic forces in two dimensions and internal flow fields. The droplets are subsequently split at a second T-junction into two daughter droplets-one containing the supernatant and the other containing the corresponding particles. The separation efficiency is measured in detail for polystyrene (PS) beads as a function of droplet speed, size, split ratio, and particle concentration. Further, single-bead (PS) and single-cell (yeast) experiments were carried out. At a throughput of 114 droplets/min, a separation efficiency of 100% ± 0% was achieved for more than 150 droplets. Finally, mammalian cells and bacteria were introduced into the system to test its versatility. This work demonstrates a robust, non-invasive strategy to perform single yeast cell-supernatant sampling in nanoliter volumes.

Acoustic Radiation Forces Produced by Sharp-Edge Structures in Microfluidic Systems.

We study sharp-edge structures that are used in microfluidic systems for particle and cell manipulation. Experiments show that oscillating sharp edges can attract or repel particles suspended in a microfluidic channel. This effect is caused by acoustic radiation forces induced by sharp edges. We propose an analytical theory that allows one to evaluate the acoustic radiation force produced by a sharp-edge structure on elastic particles and to study which parameters govern the interaction of particles with a sharp-edge structure, forcing them to be attracted in one situation and to be repelled in another situation. The proposed theory gives foundations for the design of microfluidic systems making use of sharp edges for particle trapping. We also provide experimental data to validate the theory.